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Metabolism of a Polychlorinated Piolionyl
(Aroclor50 31) Mixture in the Pal
ly D. L. Grant, \V. K. J. Phillips, and I). C. ViLi.KNr.nvn
Research f.,n}orntt/rics,
ami Dinf! Directorate
Department oj National tfmlth nnt! U t l/an;
Titnrtey's I*usturi\()ftuivu .'i. Ontario
Group3
Day 1
Polychlorinated biphenyls (PCE's) have been used in industr/ Gay 2
since at least 1930.
In 1955, von Oettingen (1) reviewed the
literature on the toxicity of these compounds.
Industrial \
workers have developed lesions of chloracne as a result 1
leaking vapours of a chlorinated biphenyl (Aroclor u{) *) used as
ray Day
3 9
a heat-exchange material (2).
Day 8
Today the problem is not restricted to exposure of industrial workers as polychlorinated biphenyls are widely dispersed in fish
Day 29
and wild life (3-11). Westoo et al. (11) found low levels of Pc?
(90%, <0.1 mg per kg fat) in a large number of foodstuffs. They
also reported that all 22 samples of human milk analyzed 'Each groi
contained PCD residues with 11 samples between 0-0.5 and the -'corn oil
other 11 between 0.6-1.0 mg per kg fat. Therefore, there is a "a 1 + 1 CO
need to study the metabolism, distribution, storage, effect on } 'a 500 mg
reproduction, etc., of PCB's in mammals. This paper will report ! orally a
on the metabolism and distribution of Aroclor 1254 in normal and yCorn oil
carbon tetrachloride-treated rats.
: A 1+1 CC
Experimental
Experiment I
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 war. 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 yibitum. The rats were killed by decapitation, blood collected, and brain, liver, heart, spleen, kidney's, testes and omental fat removed, weighed and frozen pending analyses. `Aroclor , Monsanto Company (U.S.A.), registered trade name
for polychlorinated biphenyls.
Group3 1
Group 2
Groupf^ I
d Group
i. vo n>a. Fi ft.een (.on days
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TABLE I Doping regimen lor experiment I
Groupa
Day 1 Day 2 Day 3 Day 4 Day 8 Day 24
1 Oilb PCBd Oile Killed
--
2
Oilb PCBd Oil
e Oil Killed
3'
4
CClf 4
PCBd f
CCI4
Killed
"" --
--
CClf 4
PCBd f
CCI4
f CCI4
Killed
^Each group contained five male rats, Wistar strain, av. b.w. 375 g. bCorn oil, 2 ml per kg, administered orally.
*:A 1 + 1 CCl4:corn oil solution, 2 ml per kg, administered orally. dA 500 mg per ml com oil solution of Aroclor 1254, administered
orally at 500 mg per kg. fcorn oil, 1 ml per kg, administered orally. *A 1+1 CCl4:corn oil solution, 1 ini per kg, administered orally.
TABLE II .
I
Dosing regimen for experiment II
Group3 1 Groupb 2 Groupd 3
,,d Group
4
Nil PCB C CClf
4
ccf 4
3, 8, 12, 16, 20, 24, 20, 32, 36, 40, 44, and 47
3, and 8 and PCBC on day 2
^five male rats, Wistar strain, av. b.w. 410 g, killed on day 1.
'Fifteen male rats, Wistar strain, av. b.w. 440 g, 5 killed con days 8, 12, and 47.
A 500 mg per ml corn oil solution of Aroclor 1254 administered ^orally at 500 mg per kg. cNine tale rats, Wistar strain, av. b.w. 442 g.
A 1 + 1 cebpeorn oil solution, 2 ml per kg administered orally on day 1 and 1 ml per kg on other days.
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Polychlorinated biphenyl anaVyr.er.. Tissues wore blended with
100 nil hexane and 50 y anhydrous sodium sulfate for 10 minutes.
The extract:; were filtered, concentrated to 10 ml, 3 ml of cone.
II^S04 - fuming H? SO,} (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 Vurian 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 6 3
QF-1.
The nitrogen flow rate was 120 ml per minute with column
and injection temperatures of 193 and 225C, 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. Mexanc and acetone gave equal recoveries
of Arcelor residues.
Quadruplicate analyses of a rabbit liver
gave values of 431, 438, 464, and 474 ppm. Recoveries of spiked
samples were greater than 95S.
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 v;hi.ch 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 5.320.30X
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 bruin, liver,
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spleen, blood, tustes, heart, kidney and fat of group 2 rats
(killed on day 2k) were 10, 16, 20, 21, 22, 24, 36 and 67S,
respectively, of those of group 1 (killed, on day 4).
These
values show that the PCD residues were; being cleared from the
various tissues at different rates.
TABLE III
Residues in tissues of rats orally dosed with Aroclor 1254 (500 mg/kg)
Residue found (ppm, wet tissue)
Group
1
2
34
Blood Testes Heart Spleen Kidney Brain Liver Liver^ Fat
1.9 6 a 0. 23b 0.42 0.07
3.85 * 0.46
0.25c
19.22 0.59
4. 30* 0.44
33.18* 1.35
5.62
24.16* 2.84
5. 83 0.53
62.40* 4.37
6.17
29.17 3. 44
5.82* 1.17
36.60* 4.39
--
31.14* 2.09
11.20* 1.76
57.38* 3.91
11.08
39.98* 5.91
4.01* 0.31
41.91* 3.30
5.96
115.66* 10.55
18.85* 1.65
796.47* 64.96
18.79
1868.14*166.63
--
6137.64*556.06
--
996.16* 98.58 672.66*155.12
900.46*106.16 1149.05
Mean of five values. Standard error of the `'Single value. aPpm on a fat basis.
mean.
GI.C-EC tracings from 24 ng of Aroclor 1254 and from the residue found in the liver of a group 2 rat, are shown 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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' Figure 1
' MINUTES
s
GLC-EC tracings from 20 rg of Aroclor 1250. (left) and fror, the residue found in the liver of a group 2 rat.
V
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 1 234 56
Percent 1 23 4 56
Aroclor 1254 12 25 18 17 15 13
12 25 18 17 15 13
Spleen Testes Liver Kidney Fat Brain Blood Heart
Spleen Testes Liver Kidney Fat Brain Blood Heart
Group 1
6 21 9 22 21 21
3 15 6 21 27 28
2 11
3 30 28 27
8 21 11 21 19 19
7 25 11 25 17 16
1 13 5 23 30 29
4 13 6 24 26 26
4 17 6 21 26 26
Group 3
7 25 14 19 18 17
6 23 14 19 19 19
4 21 11 21 22 21
8 22 15 18 18 17
10 29 16 21 13 12
6 25 13 20 18 18 5 22 14 22 18 18
5 27 15 18 18 17
Group 2
3 17
2 11 34 ' 32
1 19 2 11 35 32
0 25 2 18 26 28
0 18
2 14 34 32
1 19
4 20 28 28
2 19 4 12 34 29
1 20 3 12 33 31
1 17 2 8 31 29
Group 4 -
1 20 0 19 1 21 1 20 1 22 2 22 1 20
4 12 31 32 4 20 26 31 4 8 32 34 5 20 26 28 4 11 30 31 3 11 29 33 3 10 32 34
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Figure 1 and TABLE IV show that the components of" the Aroclor 1254 mixture with the shorter retention timer., 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 thee length of time following the oral dose (kidney, brain, liver and fat, group 1 vs 2) and the metabolic activity of the liver (liver and fat, group 1 vs 3).
TABLE V
Concentrations of the Aroclor 1254 residues in tissues relative to those in blood
Group |
Blood
Tes1 tes Heart
Spljeen i
Kidney 1
Brain
Liver
Fat |
1
1.00 9.81 12.33 14.08 15.89 20.40 59.17 508.24
2
1.00 10.24 13.88 13. 86 26.67
9.55 44.88 1601.57
3
1.00 8.62 16.21 9.51 14.90 10.89 206.08 233.89
4
1.00 22.48 24.68
-- 44.32 23.84 75.16 4596.20
*Phenochlor DPG, Prodelec's (France) registered trade name for polychlorinated biphenyls.
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However, since blood sample a are relatively easy to obtain they could to used for estimating the residue in other tissues, providing the investigator is aware of the limitations of the estimates.
i Experiment II
.
In Experiment I no rats received carbon tetrachloride alone, therefore experiment II was conducted to determine whether kroclor 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 8, 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 jtctrachloride 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.
1 TABLE VI
significant differences (Student Fisher's t test) found in organs weights from rats treated with Aroclor 1254 1 and untreated rats
1 Day killed
8 12 47
Organ
Probability
o oV
Spleen i Liver ; Heart kidney ; Testes
nil <0.00 5a ,b
nil _-
-
a ^Expressed as body weight,
Expressed as actual organ weight.
nil <0.005a,b <0.0 5b <0.05a <0.05a
<0.005a,b <0.05b
-
i
(
''
........... " '
''
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This agrees with results from experiments in this laboratory on rabbits treated with rcri'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 analyser, 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
Nil, killed on day 1a
Aroclor 1234 on day 2, killed on day 4d
Carbon tetrachloride on day 1, killed on day 4e
Aroclor 1254 on day^2, killed on day 8
Aroclor 1254 on day 2, killed on day 12
Lipid (%) 4.45b0.008C 6.19 0.23
13.22 1.17 8.35 0.99 7.78 0.38
Experiment II, group 1. Mean of five values. c Standard error of the mean
eExperinent 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 (PC0.005, 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 narked 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.
A number of the tissues from the rJts killed on day 47 were analyzed for PCB residues (TABLE VIII).
(0 t
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TABLE VIII
Residues in tissues o rats 45 days after being orally administered a single dose of Aroclor 1254 (500 mg/kg)
Residue Group'1
ppm, wet tissue 2
Blood Heart Kidney Brain Liver Fat
bc 0.18 0.05 2.71 0.61
3.39 0.29 4.19 0.34
16.04 3.79 397.30 41.86
Relative to Blood 2
1.00 15.06 18.83 23.28 89.11 2207.22
`
f*Group 2 of Experiment II killed on day 47.
n-lean 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 40% 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, 04, 80, 79, 78, 78, 84 and 33%
respectively in 20 days. Aroclor 1254 significantly increased
the size 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. V.'enterby for technical assistance ar. D.Ii. Coldwell and K.A. McCully for review of thi;; manuscript.
References
1. von Oettingen, W.F., Public Health Service Publication No. 414, 306 (1955) .
2. Meigs, J.W., Alboro, J.J., and Kartin, B.L., J. An. Med. Assoc. 154, 1417 (1954).
3. Risebrough, R.W., Reichc, P., Peekall, D.B., Herman, S.G., and Kirven, M.N., Mature 220, 1090 (1968).
4. Jensen, Hew Scientist 3J2, 612 (1966).
5. Holmes, D.C., Simmons, J.H., Tatton, J.O'G., Mature 216, 721
(1967).
-------
6. Holden A.V., and Marsden, K., Mature 216, 1274 (1967).
7. Koeman, J.H., Ten Hoever Do Brauw, M.C., and DeVos, R.H.,
Nature 221., 1126 (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., Bulletin Environmental Contamination and Toxicology 5, 171 (1970) .
10. Bagley, G. E., Reicnel, W. L., and Cromartie, E., J. Assoc. Offic. Anal. Chem. J53, 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, 911 (1959).
13. McLean A.E.M. and McLeah, E.K., Biochem. J. 100, 564 (1966).
14. Farber, T.M., Heider, A., Peters, E.L., Ritter,
D.L.,
Disraely, M., and Van Loon, i:.J., Ninth Annual Meeting,
Society of Toxicology, Atlanta, Georgia, March 15-19 (1970).
i 15. Cawthorr.a, M.A., Runyan, J., Scnnitt, M.V., and Green, J., Br. J. Nutr. 24, 357 (1970).
16. Tanaka, K., Fujita, S., Komatsu, F., and Tamura, H., Fukuokai Igaku-Zasshi 6, 544 (1969).1
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