CONFIDENTIAL PREPRINT

COMPARATIVE EFPECTS OF POLYCHLORINATED BIPHENYLS AND ORGANOCHLORINE
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)» triphenyls (PCT) and other related
compounds have important industrial uses and have been produced in large
volumes for many years.
ment,

Evidently some amounts reach the general environ­

Pesticide residue chemists became Interested in such materials

following a report from Sweden of their presence in wildlife tissues.1
This has been shown to interfere with the routine residue analyses of
many organochlorlne pesticides since complex patterns are produced in
EC-CLC,

2 3
*

Moreover, routine cleanup procedures fall to eliminate such

interferences unless special precautions are taken.

These materials un­

doubtedly contributed to eome of the "apparent” DDT found in biological
specimens predating usage of DDT in agriculture or public health and
constitute pert of the present background of llpoldal 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 well

documented.^

Many llpld-soluble chemicals induce various enzymes of the

hepatic endoplasmic reticulum (microsomal enzymes) including the drugmetabolizing hydroxylative 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.^
1 Presented at the 158th meeting, American Chemical Society, pesticide
Chemistry Divlolcn, September 8, 1969.
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In some situations such stimulation is beneficial as, for example, in
causing a more rapid detoxication of dleldrln and a lower residue stor­
age in the body.

The induction could likewise be detrimental because

certain toxifying reactions, such as activation of organothlophosphate
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 es the basis for the thin egg shell phenomenon and poor repro­
duction In certain birds chronically exposed to DDT, dleldrln and some
other orgnruvMorin* Insecticides.*’

Some polychlorinated biphenyl mater­

ial a havo already been reported to induce microsomal enzymes.^

The pre­

sent Investigation was undertaken to compare the induction potencies of
several types of polychlorinated biphenyls and trlphenyls to those of
various organochlorinc pesticides, and to obtain preliminary information
about tissue storage trends for such materials.

The results contribute

to our understanding of molecular characteristics required for mlcrosonml
enzyme induction and also allow some speculation about the consequences
of various types of chlorinated compounds found in biota.
METHODS
Groups 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 POT materials tested.

The test materials, Aroclovs'B)

provided by tlie 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

treated to contain 1 ppm dleldrln.

One group of rats in each experiment

was fed diet containing 50 ppm DDT and L ppm dleldrln.

The diets were

. 2 -

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fed for 15 days.

During chat period testa of drug and insecticide metab­

olism in vivo were conducted.

Later, tests of microsomal enzyme activity

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

Hexobarbltal sodium was administered at the rate of

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

The duration of deep sleep ending

with the return of the animal's righting rsflex was recorded as the
sleeping time,

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

phenyl)-phenylpliosphonothloate) and the O-demethylatlon of p-nltroanisole (PNA) were measured by incubation of the respective substrates with
the 10,000 X g supernatant of liver homogenates following the method of
Neal and DuBolS as modified by Kinoshlta e£ al,®

The ring hydroxylation

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
Residual dleldrin In adipose tissue was determined by electron
capture gas chromatography on hexane extracts of the tissue.

The adipose

tissue extracts required special cleanup to eliminate PCB interference in
the detection of dleldrin,

Thla wea accomplished by passing the extract

over a 2-stage chromatography column consisting of 1:1 MgO-Cellte (5V)
on the bottom and Florlsll containing 4% moisture (4") on the top.

After

eluting with hexane <200 ml) to bring out the PCB components, dleldrin
was recovered by eluting with 400 ml of a 1 + 4 dlchloromethane + hexane
solution.
Qualitative and semi-quantitative 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 triphenyls to
rata at rates up to 100 ppm in the diet 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
dograo with the percentage chlorine in the product.

Products with 60

parcent chlorine or more caused greater liver enlargement than equivalent
doses of the insecticide DDT.
Evidence of PC.R Induction of liver hydroxylating enzyme activity was
obtained with all enzyme tests employed.

Iiexobarbital sleep times were

markedly reduced by the FCB 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 hydroxylation, EPN degradation and demethylatIon
of p-nltroanisole, 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 Activ­
ities.

These enzyme activities were similarly affected In both sexes by

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

The products contain­

ing 607* chlorine or greater reduced dleldrln storage to levels found in
untreated control animals In our laboratory.

This activity toward diel-

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

- 4 - '

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Higher dosage rates <100 ppm vs 50 ppra in the diet) produced greeter
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 other a mixture of PCB:PCT, 60:40, containing 63%
chlorine)

v<*ro, in comparison to the PCB materials, less active in

farina of effect on the enzyme activities measured in vitro.

However,

hexobarbltal sleep time was more strongly influenced by the 42% Cl-PCT
material than by the equivalent PCB, and dieldrln metabolism was highly
stimulated by both the PCT-containlng materials (Table 4).
The Arochlor products fed and the residual components recovered
from adipoee ticsue were all examined by electron capture gas-liquid
chromatography.

Some degree of storage of each product was observed al­

though the residues 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 (Fig. 5) for 62%

Cl-PCB and its tissue residue.

The early GLC peaks (components low in

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

Peaks C and D in that residue match,

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

The 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.
SUMMARY
To summarize all the responses evaluated, hepatic microsomal enzyme
induction was minimal with the PCBs 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-

tachlor epoxide.
Microsomal enzyme induction caused by PCB materials is probably
adrUt-ive with that produced by organochlorine pesticides.^>10

Hence, if

any of the toxicological aspects of organochlorine pesticides In the
environment are related to their enzyme inducing action, the presence
of highly chlorinated biphenyls and trlphenyls would Intensify such
biological effects of the pesticides.
The very marked Influence of the highly chlorinated PCB and PCT
materials on dleldrin metabolism (which, by analogy, would affect all
other cyclodiene insecticides 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 extends our previous investigations of the structureactivity aspects of microsomal enzyme inducing agents (presented in part
St the 136th ACS meeting, Agriculture and Food Chemistry Division) and
the results fit well with our working hypotheses regarding the activities
of various bridged and unbridged diphenyl compounds.

6
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LITERATURE CXTEP

1,

Jensen, S.

2,

Reynolds,

New Sci,

3,

Risebrough, R. W., P. Reiche and H. S. Olcott.
Toxicol. 4:192-201(1969)

4,

Von Oettingen, W. F. U. S. Dept, of Health, Education and Welfare,
Public Health Service publication No. 414, 1955. The Halogenated
Aliphatic, Olefinic, Cyclic, Aromatic, and Aliphatic-Aromatic
Hydrocarbons Including the Halogenated Insecticides, Their
Toxicity and potential Dangers, p, 306-307.

5,

Street, J. C.

Ann. New York Acad, Sci.

6,

Peakall, D. B,

Nature

7,

p««ohvough, R. W., P. Reiche, D. B. Peakall, S. G. Herman, and
M. N. Kirvon. Nature 220:1098-1102(1968)

8,

Kinoohita, X. F,, J. P. Frawley and X. P. DuBols,
Pharmacol, 9^: 505 (1966)

9,

Kato, R. and J. R. Gillette.

L. M,

32:66(1966)

Bull.

Envir. Cont. Toxicol.

4^:128-143(1969)
Bull. Envir. Cont.

160:274-290(1969)

216:505(1967)

Toxicol. Appl.

J, Pharmacol. Exptl, Therap.

150:280(1965)

10, Street, J. C,, D. J. Wagstaff, and F, Mayer. Proc, 6th Interaserican
Conf, on Toxicol, and Indust. Med., Univ. Miami School of Medicine,
1968,
(in press)

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Table 1.

•

•

Food consumption, weight gain and efficiency, and Uvcr weights
of female rate treated with polychlorinated biphenyls and DDT. 1
Food
Cons.
g

Weight
Gain
g

Wt. Gain
t Food
g/100g

Liver Wt.
g/lOOg

Basal

226 ± 5.6

45 1 1.6

20 t 0.8

4.1 ♦ 0.44

Dieldrln, 1 ppm

233 * 3.3

49 ± 3.3*

21 t 1.6

4.3 i 0.27

+ DDT, 50 ppm

224 i 6.0

44 ± 2.4

20 ± 0.9

4.5 ± 0.44*

Treatment
Trial A

+ 48% Cl-PCB,

50 ppm
100 ppn

226 t 4.7
225 t 5.5

47 t 3.2
44 ±6.5

21 t 1.3
20 i 3.0

4.7 t 0.45**
4.7 t 0.46**

+ 54% Cl-PCB,

50 ppm
100 ppm

224 t 5.4
231 t 9.0

47 ± 2.0
48 ± 2.0*

21 t 0.7
21 ± 1.7

4.7 1 0.39**
5.5 1 0.46**

+ 60% Cl-PCB,

50 ppm
100 ppm

226 ± 7.1
225 t 5.5

50 t 3.1*
59 ± 2.2**

22 t 1.4*
26 t 3.0**

4.6 t 0.28**
5.3 i 0.22**

+ 68% Cl-PCB,

50 ppm
100 ppm

224 t 9.6
227 t 6.3

50 ± 5.0*
51 t 5.5*

22 t 1.8*
22 t 1.8*

5.1 ± 0.16**
5.1 t 0.46**

/PCT'
+ 65% Clj + ,, 50 ppm
\PCB, 100 ppm

228 * 8.3
232 t 7.9

43 * 3.0
49 ± 1.7*

19 ± 0.9
21 t 1.1

4.8 t 0.31**
5.2 t 0.37**

50 ppm
100 ppm

218 t 4.6
211 t 5.7

40 * 4.3
48 * 5.8*

18 t 1.8
18 ± 1.1

4.4 t 0.21
4.7 t 0.01**

Basal

237 i 3.8

39 t 2.3

16 * 2.5

4.0 t 0.31

Dieldrln, 1 ppm

234 * 6.9

40 ± 8.0*

17 1 3.0

4.3 1 0.32*

+ DDT, 50 ppm

221 t 2.6*

32 ± 2.3

15 t 1.6

4.5 ± 0.21**

+ 42% Cl-PCT,

Trial B

+ 21% Cl-PCB,

50 ppm
100 ppm

228 t 6.6
228 * 6.9

33 * 1.3
33 ± 5.5

14 * 1.7
14 t 2.5

4.1 t 0.33
4.1 + 0.56

+ 32% Cl-PCB,

50 ppm
100 ppm

225 i 4.5*
232 1 4.3

38 ± 1.7
39 ± 4.7

17 ± 1.9
17 1 1.3

4.4 i 0.52**
4.7 ± 0.44**

+ 42% Cl-PCB,

50 ppm
100 ppm

226 * 7.7
211 t 5.0**

39 ± 2.1
31 t 3.1

17 t 1.9
15 t 3.8

4.5 t 0.26**
4.6 ± 0.28**

+ 62% Cl-PCB,

50 ppm
100 ppm

216 $ 5.4**
230 t 5.3

35 t 2.5
48 ± 3.6**

16 1 1.1
20 t 1.2**

4.8 i 0.22**
5.3 i 0.33**

All groupa except the baeel received 1 ppm dieldrln.

Data are expressed

as means * S.E. for groups of five rats;
Asterisks, * and **, Indicate values Judged significantly different from
the basal group by LSD analysis with P S .05 or .01, respectively.
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Table 2.

Enzyme raaponsea and reduction In hexobarbltal sleep time and
dleldrln storage In female rats treated ulth polychlorinatedbiphenyls, -triphenyls or DDT. *

Treatment

EPN
Detox.
%

PNA
Demeth.
7.

Aniline
Oxidn.
7.

94

98

104

Hexobarb.
Sleep Time
-7.

Dleldrln
Storage
-7.

Trial A
Basal
+ Dleldrln, 1 ppm

100

100

100

0

0

+ DDT, 50 ppm

431

228

205

75.0

93.4

+ 48% Cl-PCB,

50 ppm
100 ppm

225
362

408
568

193
295

34.6
57.7

60.7
77.5

+ 54% Cl-PCB,

50 ppm
100 ppm

444
475

555
672

309
346

65.4
80.0

85.8
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

Trial B
Basal

92

114

114

+ Dleldrln, 1 ppm

100

100

100

0

0

+ DDT, 50 ppm

450

300

248

78.6

92.2

+ 21% Cl-PCB,

50 ppm
100 ppm

117
117

148
128

138
154

11.3
31.0

7.8
10.9

+ 32% Cl-PCB,

50 ppm
100 ppm

183
233

257
411

194
225

48.8
55.4

29.7
45.3

+ 42% Cl-PCB,

50 ppm
100 ppm

200
275

386
474

250
305

37.5
65.0

37.5
64.7

+ 62% Cl-PCB,

50 ppm
100 ppm

625
717

403
474

293
312

85.7
87.5

94.5
96.5

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 dieldrin.

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Table 3.

Sex comparisons in selected response of rats to
various polychlorinated biphenyl materials. 1

PCB Treatment

Sex

EPN
Detox.
7.

PNA
Demeth.
7.

Dleldrin
Storage
-7.

48% Cl, 30 ppm
25 ppm

P
M

193
155

416
265

46.0
46.7

547. Cl, 50 ppm
25 ppui

F
M

293
225

497
341

74.7
76.6

607. 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

687 Cl, 50 ppm
25 ppm

F
M

467
430

254
359

92.7
88.7

~*“r* •—............ ..........
Data from rats fed diets containing l ppm dleldrin plus
each treatment at the indicated level. Each value Is the
mean percentage Increase (or decrease) In the measured re­
sponse of groups of five rats compared to groups that receiv­
ed the basal diet containing only 1 ppm dleldrin.

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

Tablo 4

EPN
Detox.
7.

PNA
Demeth.
7.

Aniline
Oxidn.
7

Hexobarb.
Sleep Time
-7.

Dleldrin
Storage
-7

427. Cl-PCB
427 Cl-PCT

200
238

386
208

250
145

37.5
46.2

37.5
73.6

687. Cl-PCB
627 Cl-PCB
657 C1-, PCB

600
625
519

272
403
249

266
293
231

85.6
85.7
66.3

96.4
95.0
95.5

Treatment

, PCT
1 Data from female rats fed diets containing 1 ppm dleldrin plus each
treatment at a level of 30 ppm. Each value is the mean percentage in­
crease (or decrease) in the measured response of groups of five rats com­
pared to groupo that received the basal diet containing only l ppm
dleldrin.

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Figure 1. Reduction In hexobarbltal 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-nltroanlsole after receiving FCB
materials in the diet (SO ppm) for IS days.
Data are presented as percentage increase
In activity relative to rata receiving the
basal diet containing, only 1 ppm dleldrln.

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o

Figure 3. Reduction In dleldrin storage
In adipose tissue of female rats fed
diets containing 1 ppm dleldrin and
various PCB materials (50 ppm) for 15 days.

IN DIET

Figure 4. Dose response curves for various
nlcro8omal enzyme Inducing agents In terms
of stimulation of the in vitro degradation
of EPN by liver mitochondrial supernatant
preparations. The inducing agents were ad­
ministered in the diets of female rats for
15 days.

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

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