'CONFIDENTIAL PREPRINT

COMPARATIVE EFFECTS OF POLYCHLORINATED BIPHENYLS AND ORGAHOCHLORINE
PESTICIDES IN INDUCTION OF HEPATIC MICROSOMAL ENZYMES1
Joseph C. Street, Francis M, Urry,
D* Jesse Wagstaff and Adrian D. Blau
Department of Animal Science
Utah State University
Logan, Utah 84321

Chlorinated biphenyls (PCB) , trlphenyls (PCT) and other related
compounds have important Industrial uses and have been produced In large
vnitimeit for many years.

meat.

Evidently some amounts reach the general environ*

Pesticide residue chemists b«c*m« interested in such materials

following a report from Sweden of their presence in wildlife tissues.^
This has been shown to Interfere with the routine residue analyses of
many organochlorlne pesticides since complex patterns are produced in
2 3
EC-GLC. ’

Moreover, routine cleanup procedures fail to eliminate auch

lntarforencea unless special precautions are taken.

These materials un­

doubtedly contributed to soma of the "apparent” DDT found in biological
specimens predating usage of DDT in agriculture or public health and
constitute part of the present background of lipoldal organic chlorine
In the biota.
Polychlorinated biphenyls produce acute toxic effects in mammals
similar to those of chlorinated naphthalenes and other chlorinated aro­
matics.

The special vulnerability of the liver to such agents is wsll

documented.^

Many lipid-soluble chemicals induce various enzymes of the

hepatic endoplasmic reticulum (microsomal enzymes) including the drugmetabolizing hydroxylotive system.

Several organochlorlne pesticides are

potent inducers of such enzymes, with the result that treated animals
metabolize drugs, insecticides and other foreign compounds,more rapidly.5
* Presented at the 158th meeting, American Chemical Society, Pesticide
OhemUtry Division, September 8, 1969.
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034627

In soma situations such stimulation Is beneficial as, for example, In
causing a more rapid detoxication of dieldrln and a lower residue stor­
age In the body.

The induction could likewise be detrimental because

certain toxlfylng reactions, such as activation of organothiophosphate
insecticides, are also stimulated.

Enhanced steroid degradation also

occurs which raises possibilities of disturbing certain physiological
processes due to altered endocrine relationships.

The latter has been

suggested as the basis for the thin egg shell phenomenon and poor repro­
duction In certain birds chronically exposed to DDT, dieldrln end soma
other orgsnorhlorlna Insecticides.*

Some polychlorinated biphenyl mater­

ials havo already been reported to induce microsomal enzymes.^

The pre-

sont Investigation was undertaken to compare the Induction potencies of
several types of polychlorinated biphenyls and trlphenyls to those of
various organochlorlne pesticides, and to obtain preliminary Information about tissue storage trends for such materials.

The results contribute

to our understanding of molecular characteristics required for microsomal
enzyme induction and also allow some speculation about the consequences
of various types of chlorinated compounds found In biota.
METHODS
Croups of male and female rats were Individually fed Purina Labora­
tory Chow diets containing 25, 50, and 100 ppm concentrations of one of
the several PCB and PCT materials tested.

The test material's, Aroclors®

provided by the Monsanto Co., consist of crude products obtained by
chlorination of biphenyl, or triphenyl, to specified limits; each Is
therefore a complex mixture.
cent chlorine.

The ten products ranged from 21 to 68 per­

Except for the negative controls, the diets were also

troated to contain 1 ppm dieldrln.

One group of rats In each experiment

was fed diet containing 50 ppm DDT and 1 ppm dieldrln.

The diets were

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£ed for IS days.

During that period tests of drug and insecticide metab­

olism in vivo were conducted.

Later, tests of microsomal enzyme activity

were carried out with liver homogenates,
Hexobarbital sleeping time measurements were made on the tenth
day of treatment.

Hexobarbital sodium was administered at the rata of

100 mg/kg l.p. in aqueous solution.

The duration of deep sleep ending

with the return of the animal's righting reflex was recorded as the
sleeping time.

The oxidative detoxification of EFN (0-ethyl-0-(p-nitro-

phenyl)-ph(tnylphosphonothloate) and the O-demethylation of p-nitroanlsole (PHA) were measured by incubation of the respective substrates with
the 10,000 X g supernatant of liver homogenates following the method of
Heal and DuBols as modified by Klnoshlta et_ el.

O

The ring hydroxy laden

of aniline was measured by the incubation of aniline hydrochloride with
the 10,000 X g supernatant of liver homogenates following the method of

'

Kato and Gillette.9
Xesldual dieldrin in adipose tissue was determined by electron
capture gaa chromatography on hexane extracts of the tissue.

The adipose

tissue extracts required special cleanup to eliminate PCB Interference in
the detection of dieldrin.

This was accomplished by passing the extract

over a 2-stage chromatography column consisting of 1:1 MgO-Celite (SV1)
on the bottom and Florlsll containing 4% moisture (4") on the top.

After

eluting with hexane (200 ml) to bring out the PCB components, dieldrin
was recovered by eluting with 400 ml of a 1 + 4 dichloromethane + hexane
solution.

.

Qualitative and seml-quantltatlve evaluation of PCB storage In
adipose tissue was possible after careful evaluation of GLC data comparing
parent material and that recovered from tissue.

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RESULTS
Short term feeding of polychlorinated biphenyls and trlphenyls to
rats at rates up to 100 ppm In the diat had essentially no effects on
food consumption, weight gains or efficiency of food utilization (Table 1).
Liver enlargement was observed with all products tested, Increasing In
dogree with the percentage chlorine In the product.

Products with 60

percent chlorine or more caused greater liver enlargement than equivalent
doses of the insecticide DDT.
Evldsnca of FOB Induction of liver hydroxylatlng enzyme activity was
obtained with all enzyme tests employed.

Hexobarbital sleep times were

markedly reduced by tha PCB materials at both SO and 100 ppm In the diet
for 10 days (Fig. 1)•

The rate of decrease was reasonably linear with

increasing degree of PCB chlorination.
Enzymatic aniline hydroxylatlon, EFN degradation and demathylatIon ’
of p-nltroanlsole, as determined with liver homogenates, were Increased
with preparations from PCB-treated rats (Fig. 2 and Table 2).

In general,

higher chlorination of the biphenyl resulted In greater enzymatic activltlas.

These enzyme activities were similarly affected In both sexes by

tha treatment materials, although the male appeared to be more responsive
(Table 3) .
Dleldrln metabolism In vivo, judging from Its residue storage In
adipose tissue, was stimulated by tha treatment materials, especially
those with high degrees of chlorination (Fig. 3).

The products contain­

ing 602 chlorine or greater reduced dleldrln storage to levels found In
untreated control animals in our laboratory.

This activity toward dlel­

drln metabolism had been predicted by analogy to the similar effects of
anzyma-lnduclng drugs and organochlorlne pesticides.

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Higher dosage ratea (100 ppm vs 50 ppm In the diet) produced greater
degrees of enzyme Induction.

The dose-response curve (Fig. 4) obtained

with 62% Cl-PCB, in the range from 5 ppm to 100 ppm in the diet, was
generally similar to that of DDT or phenobarbital yet that substance was
greater in potency at every dosage level,

Heptachlor epoxide, by contrast,

was considerably more potent than any of these compounds.
The two products containing chlorinated triphenyl components (one
42% Cl-PCT and the ocher a mixture of PCB:PCT, 60:40, containing 65%
chlorln*) tasted veto, in comparison to the PCB materials, leas active In
terms of effect on the enzyme activities measured In vitro.

However,

hexobarbital sleep time was more strongly influenced by the 42% Cl-PCT
meterial than by the equivalent PCB, and dleldrln metabolism was highly
stimulated by both the PCT-contalnlng materials (Table 4).
The Arochlor products fed and the residual components recoverad
from adipose tissue were ell examined by electron capture gas-liquid
chromatography.

Soma degree of storage of each product was observed al­

though the reeldues had greatly modified composition in comparison to the
parent materials administered.

Those products and Individual components

having the least chlorine appeared to be stored the least In adipose
tissue.

That can be seen In the comparison Illustrated (Pig. 5) for 62%

Cl-PCB end its tissue residue.

The early GLC peeks (components low In

chlorine) In the residue were greatly diminished relative to moat of the
later peaks in the chromatogram.

Peeks C and D In that residue match,

In retention time, two of the three major peaks reported by Risebrough
at .1.3 in PCB residues from tissues and eggs of birds in the San Fran­
cisco bay area,

Tha tissue residues for PCBs lower In chlorination were

much lower in proportion to that of the highly chlorinated PCBs and

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'

consisted chiefly of minor components of those materials*

Work Is In

progress to quantitate these residues in terms of organic chlorine,
SU^RY
To summarize all the responses evaluated, hepatic microsomal enzyme
induction was minimal with the PCBa of low chlorination <21-32% chlorine)
but Increased to very high values for those materials averaging over 60%
chlorine.

The latter exceeded DDT in potency of induction, but not hep*

techlor epoxide,

-

'

Microsomal, anzyroo induction caused by PCB materials is probably
6 10

additive with that produced by organochlorlne pesticides, ,L

Hence, if

any of the toxicological aspects of organochlorlne pesticides in the
environment are related to their ensyme inducing action, the presence
of highly chlorinated blphenyla and trlphanyls would Intensify such
biological effects of the pesticides.
The very marked influence of the highly chlorinated PCB and per
materials on dleldrln metabolism (which, by analogy, would.affect all
other cyclodiene lniactlcldee and DDT as well) could greatly confound the
interpretation of exposures of animals and birds bearing mixed residues.
The toxicological implications of such exposures would be equally con­
founded.
This work axtenda our previous investigations of the structureactivity aapacts of microsomal enzyme inducing agents (presented in part
at tha 136th ACS meeting, Agriculture and Food Chemistry Division) and
the results fit well with our working hypotheses regarding tha activities
of various bridged and unbrldged diphenyl compounds.

HONS
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LITERATURE CITED

1.

Jensen, S.

2.

Reynolds, L. M.

New Scl,

32^66(1966)

3.

Rlsebrough, R, W., p. Relche and H. S. Olcott.
Toxicol. 4:192-201(1969)

4.

Von Oettlngen, W. F. U. S. Dept, of Health, Education and Welfare,
Public Health Service Publication No. 414, 1933. The Halogenated
Aliphatic, Oleflnlc, Cyclic, Aromatic, and Aliphatic-Aromatic
Hydrocarbons Including the Halogenated Insecticides, Their
Toxicity and Potential Dangers, p. 306-307.

Bull. Envlr. Cont. Toxicol.

4:128-143(1969)
Bull. Envlr. Cont.

5.

Street, J. C,

Ann. New York Acad. Scl.

6.

Peakall, D. B.

Nature

7.

pf.cbroueh, R. W., P. Relche, S. B. Peakall, S. G. Herman, and

M. N. Klv-von.

160:274-290(1969)

216:505(1967)

Nature

220:1098-1102(1968)

8.

Klnoshlta, K. F., J. P. Frawley and K. P. DuBols.
Pharmacol. 9:505(1966)

9.

Kato, R. and J. R. Gillette.

10.

Street, J, C., D. J. Wagataff, and F. Mayer, Proc. 6th Interamerlcan
Conf. on Toxicol, and Indust. Med., Unlv. Miami School of Medicine,
1968,
(in press)
-

J. Pharmacol. Exptl. Therap.

MQNS

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Toxicol. Appl.

034633

150:280(1963)

Tabl« 1.

Food consumption, weight gain and efficiency, . id liver weights
of female rats treated with polychlorinated biphenyls end DDT. *■
Food
Cons.
8

Treatment

Height
Gain
8

Wt. Cain
C Food
g/lOOg

Liver Wt.
g/lOOg

Trial A
Basel

226 ± 5.6

45 1 1.6

20 a 0.8

4.1 a 0.44

Dieldrln, 1 ppm

233 ± 3.3

49 i 3.3*

21 i 1.6

4.3 a 0.27

4 DDT, 50 ppm

224 1 6.0

44 1 2.4

20 a 0.9

4.5 a 0,44*

+ 48* Cl-PCB,

50 ppm
100 ppm

226 1 4.7
225 1 5.5

47 ± 3.2
44 1 6.5

21 a 1.3
20 a 3.o

4.7 a 0.45**
4.7 a 0.46**

+ 54* Cl-PCB,

50 ppm
100 ppm

224 ± 5.4
231 ± 9.0

47 a 2.0
48 1 2.0*

21 ± 0.7
21 a 1.7

4.7 a 0.39**
5.5 a 0.46**

4 60* Cl-PCB,

50 ppm
100 ppm

226 a 7.1
225 t 5.5

50 a 3.1*
59 1 2.2**

22 a i.4*
26 a 3.0**

4.6 a 0.28**
5.3 a 0.22**

4 68* Cl-PCB,

50 ppm
100 ppm

224 ± 9.6
227 1 6.3

50 a 5.0*
51 1 5.5*

22 a 1.8*
22 a 1.8*

5.1 a 0.16**
5.1 a 0.46**

ipcr\
4_ ,, 50 ppm
\PCB 100 ppm

228 1 8.3
232 f 7.9

43 i 3.0
49 i 1.7*

19 a o.9
21 a l.i

4.8 a 0.31**
5.2 a 0.37**

50 ppm
100 ppm

218 1 4.6
211 i 5.7

40 1 4.3
48 1 5.8*

18 * 1.8
18 a l.i

4.4 a 0.21
4.7 a o.oi**

Basal

237 1 3.8

39 t 2.3

16 a 2.5

4.0 a 0.31

Dieldrln, 1 ppm

(•*>
CM

t 6.9

40 a 8.0*

17 a 3.0

4.3 a 0.32*

4 DDT, 50 ppm

221 1 2.6*

32 a 2.3

15 a 1.6

4.5 a o.2i**

4 21* Cl-PCB,

50 ppm
228 t 6.6
100 ppm . 228 t 6.9

33 a 1.3
33 a 5.5

14 a 1.7
14 a 2.5

4.1 a 0,33
4.1 a 0.56

4 32* Cl-PCB,

50 ppm
100 ppm

225 a 4.5*
232 t 4.3

38 a 1.7
39 ± 4.7

17 a 1.9
17 a 1.3

4.4 a 0.52**
4.7 a 0.44**

4 42* Cl-PCB,

50 ppm
100 ppm

226 1 7.7
211 i 5.0**

39 a 2.1
31 a 3.1

17 a 1.9
15 a 3.8

4.5 a 0.26**
4.6 a 0.28**

4 62* Cl-PCB,

50 ppm
100 ppm

216 ± 5.4**
230 a 5.3

16 a 1.1
35 *2.5
48 a 3.6** _20 a 1.2**

4 65* Cl

4 42* Cl-PCI,

Trial B

<

4.8 a 0.22**
5.3 a 0.33**

All groups except the basal received 1 ppm dieldrln. Data are expressed
as means 1 S.E. for groups of five rats.
Asterisks, * and **, Indicate values Judged significantly different from
the basal group by LSD analysis with P % .05 or .01, respectively.

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Table 2.

Enzyme raspons-s and reduction in hexobarbltal -eep time and
dleldrin storage in female rata treated with polychlorinatedbiphenyls, -triphenyls or DDT. 1

Hexobarb,
Sleep Time
-7.

Dleldrin
Storage
-%

0

0

205

75.0

93.4

193
295

34.6
57.7

60.7
77.5

EPN
Detox.
%

PNA
Demeth,
%

Aniline
Oxidn*
%

Basal

94

98

104

+ Dleldrin, 1 ppm

100

100

100

4- DDT, 50 ppm

431

228

Treatment
Trial A

50 ppm
100 ppm

225
362

408
568

+ 54% Cl-PCB,

50 ppm
100 ppm

444
475

555
672

309
346

65.4
80.0

85. B
91.6

+ 60% Cl-PCB,

50 ppm
100 ppm

475
544

291
368

231
273

78.8
80.0

93.4
96.5

+ 68% Cl-PCB,

50 ppm
100 ppm

600
525

272
242

266
240

83.7
80.0

96.5
96.0

Basel

92

114

114

4- Dleldrin, 1 ppm

100

100

100

0

0

4- DDT, 50 ppm

+ 48% Cl-PCR,

.

Trial B

450

300

248

78.6

92.2

4- 21% Cl-PCB, 50 ppm
100 ppm

117
117

148
128

138
. 154

11.3
31.0

7.8
10.9

4- 32% Cl-PCB,

50 ppm
100 ppm

183
233

257
411

194
225

48.8
55.4

29.7
45.3

4- 42% Cl-PCB,

50 ppm
100 ppm

200
275

386
474

250
305

37.5
65.0

37.5
64.7

4- 62% Cl-PCB,

50 ppm
100 ppm

625
717

403
474

293
312

85.7
87.5

94.5
96.5

.

1 Each value is the mean percentage increase (or decrease) in the measured
response for groups of five rats compared to the groups that received the
basal diet plus only 1 ppm dleldrin.

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Table 3,

Sex comparison* In selected response of rats to
various polychlorinated biphenyl materials. 1

PCB Treatment

■ Sex.

EPN
Detox.
7.

PKA
Demeth.
7.

Dleldrln
Storage
-X

487. Cl, 50 ppm
25 ppm

F
M

193
155

416
265

46.0
46.7

54% Cl, 50 ppm
25 ppm

F
M

293
225

497
341

74.7
76.6

60% Cl, 50 ppm
25 ppm

F
M

413
340

316
331

91.8
85.0

62% Cl, 50 ppm
25 ppm

F
M

433
408

286
363

91.5
90.0

68% Cl, 50 ppm
25 ppm

F
M

467
430

254
359

92.7
88.7

* Data from rata fed diets containing 1 ppm dleldrln plus
each treatment at the Indicated level. Each value Is the
mean percentage Increase (or decrease) In the measured re­
sponse of groups of flva rats compared to groups that receiv­
ed the basal diet containing only 1 ppm dleldrln.

Table 4,

Comparisons of responses from polychlorinated biphenyls to poly­
chlorinated triphenyl materials equivalent In chlorination.*

Treatment

42% Cl-PCB
42% Cl-PCT
68% Cl-PCB
62% Cl-PCB
65% Cl-/ PCB)
I + '

.

EPN
Detox.
%

PNA
Demeth.
%

Anlllna
Oxidn,
%

Hexobarb.
Sleep Time
-7.

Dleldrln
Storage

200
238

386
208

250
145

37,5
46.2

37.5
73.6

600
625
519

272
403
249

266
293
231

85.6
85.7
66.3

■

-in"

96.4
95.0
95.5

PCT

* Data from female rats fed diets containing 1 ppm dleldrln plus each
treatment at a level of SO ppm. Each value is the mean percentage In­
crease (or decrease) in the measured response of groups of five rats com­
pared to groups that received the basal diet containing only .1 ppm
dialdrin.

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034636

Figure 1. Reduction in hexobarbital sleep
time in female rats after receiving FCB
materials In the diet (SO ppm) for 10 days.

Figure 2.
Increases In activity of liver
homogenates from female rats In degrading
EPN and p-nitroanlsole after receiving PCB
materials In tha diet (SO ppm) for IS days,
Data are presented as percentage increase
In activity relative to rats receiving the
basal diet containing only 1 ppm dleldrln.

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I.

1> CM.0NM1I0H Of 1HE BIPHEKYl

Figure 3, Reducdon in dleldrln storage
In adipose tissue of female rats fed
diets containing 1 ppm dleldrln and
various PCB materials (SO ppm) for IS days.

Figure 4. Dose response curves for various
microsomal enzyme Inducing agents In terms
of stimulation of the In vitro degradation
of EPN by liver mitochondrial supernatant
preparations. The Inducing agents vere ad­
ministered In tha diets of female rats for
IS days.
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I

Figure 5.
Electron capture gas chromatograms
of 62% Cl-PCB material and Its residue recovered
from rat adipose tissue.

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034639