[CANCER RESEARCH 35. 1574-1579,June 1975]
Effect of Polychlorinated Biphenyls (Aroclor 1254) on Inducible and
Repressible Microsomal N-Demethylases in the Mouse and Rat'
Mary F. Argus, Georgia M. Bryant, Karen M. Pastor, and Joseph C. Arcos2
Seamen's Memorial Research Laboratory, USPHS Hospital, 210 State Street, New Orleans, Louisiana 70188; and Department of Medicine,
Tulane University Medical Center, New Orleans,Louisiana
SUMMARY
de novo synthesis of the enzyme (2, 35, 36) and not to
inhibition ofthe activity ofexisting enzyme by the hydrocar
A comparative study of the effects of the polychlorinated
bon or its metabolite(s) (33). Consistent with the well-estab
biphenyl mixture Aroclor 1254, 3-methylcholanthrene,
and
lished requirement of demethylation for the toxicity of and
starvation on hepatic dimethylnitrosamine
(DMN) demeth
carcinogenesis by DMN are the findings that the repression
ylase (a repressible enzyme) and azo dye N-demethylase (an of hepatic DMN demethylation by MC (3, 5, 29, 33, 36) is
inducible enzyme) has been carried out. As previously
paralleled by the decrease of toxicity of (33) and inhibition
observed with polycyclic hydrocarbons and phenobarbital,
of hepatocarcinogenicity
of (20) DMN owing to MC
Aroclor in rats is a potent inducer of liver tissue prolifera
administration
and That the substantial lowering of DMN
tion and of azo dye N-demethylase,
as well as a @iotent metabolism by aminoacetonitrile
(2, 3, 15, 19) is paralleled
repressor of DMN demethylase.
However, in mice, al
by the inhibition of DMN toxicity (18, 19) and carcinogene
though the inducing effect on liver tissue proliferation and sis (17, 19) by aminoacetonitrile.
Similarly, Dibenamine
azo dye N-demethylase activity is maintained, there is no [N-(2-chloroethyl)dibenzylamine],
which substantially de
change in DMN demethylase activity as a result of Aroclor
creases DMN demethylation (3 1), also reduces the hepato
administration.
As in rats, 3-methylcholanthrene
induces
toxicity of DMN (24), as well as the hepatocarcinogenicity
the azo dye N-demethylase
in mice. This hydrocarbon,
of diethylnitrosamine
(37).
In contrast
to the effect
of these
which is known to substantially repress the DMN demethyl
chemical agents, the dietary control of the DMN demethyl
ase in rats, has, however, no effect on this enzyme in mice.
ase is similar to the dietary regulation of a number of other
While starvation is known to have a substantial inducing
enzymes in that it is induced by amino acids and repressed
effect on DMN demethylase in rats, in mice starvation
by carbohydrates (34).
brings about a moderate induction of DMN demethylase.
Since all the foregoing studies were carried out in rats, a
recent paper by Czygan et a!. (14) raised intriguing possibili
ties. These investigators, working with mice, reported that
INTRODUCTION
the PCB mixture, Aroclor 1254, is a powerful inducer of the
demethylation of DMN in the liver. Aroclor 1254 is known
The enzyme system catalyzing the demethylation of to be an inducer of hepatic benzo (a)pyrene hydroxylase (1),
DMN,3 referred to as DMN demethylase, is an unusual aniline hydroxylase, and ethylmorphine N-demethylase (6),
mixed-function
oxidase. Previous investigations
showed
as well as of the metabolism of zoxazolamine and hexobar
that while it requires NADPH and oxygen (33) like all bital (6). It is well established that among these mixed-func
typical mixed-function
oxidases, pretreatment
of the ani
tion oxidases, benzo (a) pyrene hydroxylase, aniline hydrox
mals with enzyme inducers, such as polycylic aromatic
ylase, and zoxazolamine hydroxylase are also induced by
hydrocarbons
and phenobarbital
(3, 5, 29, 33, 36) and polynuclear hydrocarbons. In view of these observations the
pregnenolone- 16a-carbonitrile, aminoacetonitrile, and fi possibility was at hand either that Aroclor 1254 is a unique
naphthoflavone (2, 3, 15, 19, 29, 30), brings about decrease
agent that induces DMN demethylase synthesis and/or that
of enzyme activity. It has been established that the decrease
the response of this enzyme system to pretreatment of the
brought about by hydrocarbons is due to repression4 of the animals by enzyme inducers is diametrically opposite in rats
1 This
investigation
CA-13206
from
was
supported
the National
by
Cancer
USPHS
Research
Institute.
Financial
Grant
aid from
of de novo synthesis. Designation
of the observed decrease of DMN
demethylaseactivity due to MC pretreatmentas repressionis basedon the
Hoffmann LaRoche Inc., through the kindnessof Dr. Allan H. Conney,is
following
gratefully acknowledged.
apparent Km but does causesignificant decreaseof the Vmaz(36); (b) in
2 Recipient
of
a
Faculty
Research
Award
from
the
American
Cancer
Society.
3 The
abbreviations
nitrosodimethylamine);
biphenyl; 3-Me-MAB,
4 The
term
used
are:
DMN,
dimethylnitrosamine
(N-
MC, 3-methylcholanthrene; PCB, polychlorinated
3-methyl-4-monomethylaminoazobenzene.
“repression―
is
used
here
to
mean
the
counterpart
of
true
enzymeinduction, i.e., increasedamount ofenzyme dueto acceleratedrate
Received July 26. 1974; accepted March 11, 1975.
1574
findings: (a) MC pretreatment
brings about no change in the
vitro added MC, unchanged or after metabolism, has no effect on the rate
of demethylation of DMN (33) indicating that the decreaseof Vmax@5
not
due to noncompetitiveinhibition; (c) MC doesnot inducethe synthesisof
an endogenous inhibitor of DMN demethylase (36); (d) investigation of the
2 possible alternatives that can account for the decrease of the Vms,,
decreased de novo synthesis or increased enzyme catabolism, ruled out the
decreaseof DMN demethylasehalf-life by MC pretreatment (2). A full
report of the latter studies has been submitted for publication.
CANCER
RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
Microsomal
N-Demethylases
in the Mouse and Rat
enzyme) in rats and mice.
ing point of 89—90°
[literature
3-methyl-4-aminoazobenzene,
dard curve for estimation of
product formed in the enzyme
MATERIALS
A. Miller and Dr. Elizabeth C. Miller. The protein content
of the microsomal and 9000 x g postmitochondrial
prepara
tions were determined by the method of Lowry el al. (22).
versus mice. Hence this report describes a comparative
study of the effect of Aroclor 1254 on the DMN demethyl
ase and the azo dye N-demethylase
(a typical inducible
AND METHODS
Male Swiss-Webster mice (Camm Research Institute,
Inc., Wayne, N. i.)5 and male Sprague-Dawley rats (The
Holtzman Co., Madison, Wis.) were used. The mice were
maintained on Purina laboratory chow and the rats, as
previously, on an 18% casein diet containing here 4 mg
riboflavin/kg (cf Refs. 33 and 36), given ad libitum. At the
time of sacrifice the weight range of the mice was 20 to 26 g
and the weight range of rats was 75 to 90 g. Aroclor 1254
(provided
by William
B. Papageorge,
Monsanto
Co., St. Louis, Mo.) and MC (Eastman
Chemical
Kodak Co.,
Rochester,
N. Y.) were administered
by a single i.p.
injection in corn oil at the respective levels of 500 and 40
mg/kg body weight; in some experiments MC was given at
the level of 80 or 160 mg/kg body weight. Controls received
the same volume of corn oil only. The animals were always
sacrificed between 8 and 9 a.m. to avoid the possible
influence of circadian variations.
For the DMN demethylase assay the method of isolation
of
the
microsomes,
the
demethylation
reaction,
and
the
formaldehyde determinations
were as previously described
(33, 36), except that in this study the final volume of the
demethylation
medium as well as all components were
halved.
The azo dye N-demethylase assay was carried out on a
9000 x g postmitochondrial supernatant fraction of a 20%
homogenate in 1. 15% KCI. The demethylation reaction was
conducted in the assay system of Conney et a!. (13), as
modified by Bresnick and Stevenson (7). For the measure
ment of the demethylation of 3-Me-MAB the metabolites
were separated by thin-layer chromatography
and estimated
on eluates of the spots by the method of Bresnick and
Stevenson (7). In our experiments the ratio of the RF of
3-Me-MAB
over the RF of the demethylated
3-methyl-4-aminoazobenzene,
product,
was 2.00 ±0.01 with rats
and 2.06 ±0.01 with mice; this ratio was 2.04 in the study
of Bresnick and Stevenson (7).
3-Me-MAB used as substrate for the azo dye N-demeth
ylase assay was prepared by the method of Miller and
Miller (25), by coupling benzenediazonium
chloride to
N-methyl-o-toluidine
followed by rearrangement
of the
resulting 2-methyl-N-phenylazo-N-methylaniline
to 3-Me
MAB. The crude 3-Me-MAB hydrochloride was submitted
to 15 to 20 trituration in and extraction with, ice-cold 10%
hydrochloric acid, before decomposition with excess alkali
and crystallization
of the free base in benzene-petroleum
ether 40—60°.A 3-times-crystallized
sample had a melt
(25), 89—90°].
The sample of
used to establish the stan
the amount of demethylated
assay, was a gift of Dr. James
RESULTS
Effect
of Aroclor
1254 on Hepatic
Tissue
Synthesis.
Table 1 presents the effect of this PCB, as well as of starva
tion, in rats and mice [the administration
schedule of the
experiment summarized in Table 1, Row 3, was designed to
specifically conform to the conditions of Czygan et al. (14)].
Table 1 shows that pretreatment with Aroclor brings about
a substantial increase in the liver weight/body weight ratio
in both rats and mice, provided a 4-day period is allowed
between administration
and sacrifice (Table 1, Rows 1
and 2); moreover, with mice, this ratio appears to undergo a
larger increase if the experiment is carried out on animals
submitted to a 12-hr starvation period (Table 1, Row 3).
If the 4-day period between Aroclor administration
and
sacrifice is reduced to 24 hr, liver hypertrophy is considera
bly decreased (Table 1, Row 4).
Unlike the liver weight/body
weight ratio, which is
increased in both rats and mice by Aroclor, the response of
the 2 species is different in terms of microsomal yield per
unit weight of tissue. While rats show a substantial decrease
(Table 1, Row 1), mice, under an identical administration
schedule (Table I, Row 2), show no change. However, if
the experiment is carried out on mice submitted to a 12-hr
starvation
period, a statistically
significant increase is
observed (Table I, Row 3).
Starvation of mice for 24 hr (in absence of Aroclor)
brings about a decrease of both the liver weight/body weight
ratio and the microsomal yield (Table I, Row 5).
In contrast to the effect of Aroclor, pretreatment with
MC does not increase the liver weight/body weight ratio of
mice. Not tabulated results show that, 24 hr following
administration
of MC, 40 mg/kg body weight, the ratios
were: control, 6.41 ±0. 11; experimental, 6.29 ±0. 17 (0.60
> p > 0.50). Similarly, the microsomal yield per unit
weight of tissue remains unchanged: control, 11.38 ±0.43;
experimental, 1137 ±1.08 (0.20 > p > 0. 10). There is no
change in the ratio or in the microsomal yield by increasing
the MC dose to 80 or 160 mg/kg.
Effect of Aroclor 1254 on Hepatic DMN Demethylase.
Enzyme activity in Table I is expressed in terms of
formaldehyde produced per both mg microsomal protein
and g of tissue. In rats pretreatment
with Aroclor brings
about a very substantial
decrease of enzyme activity,
expressed either way. In surprising contrast, in mice hepatic
DMN demethylase remains unaffected by Aroclor pretreat
5 This
is the same
source
for this strain
of mice
as used for the study
by
Czygan et al. ( 14) who erroneously indicated that the mice were purchased
from the “Cancer
Research Institute, Wayne, N. J.―The Swiss-Webster
mice used by Czygan et al. were actually purchased from the Camm
Research Institute, Inc., Wayne, N. J. (D. Y. Cooper, personal communi
cation).
JUNE
ment. The 23.1% increase (as zmoles HCHO per hr per g
tissue) in Table 1, Row 3, reflects actually the increase of
the amount of microsomes (22.5%) rather than an actual
selective induction of DMN demethylase synthesis, since
enzyme activity expressed per g tissue is the product of the
1975
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
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CANCER RESEARCH VOL. 35
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
Microsoma!
N-Demethy!ases
in the Mouse and Rat
microsomal yield (in terms of mg microsomal protein per g
Table 2, Row 4; and 5.01 ±1.40 for Table 2, Row 5. The
liver) and of the enzyme activity (expressed per mg micro
ratio was I .00 for Table 2, Row 6, indicating that starvation
did not bring about stimulation of polar metabolite forma
tion in mice.
somal protein). The small decrease in Table I, Row 4, is
not significant statistically.
Starvation
of mice for 24 hr (in absence of Aroclor) has a
moderate but clearly detectable inducing effect on DMN
demethylase, expressed as HCHO produced per hr per mg
microsomal
DISCUSSION
protein. If enzyme activity is expressed per g of
Aroclor 1254, a potent inducer ofvarious mixed-function
tissue, however, there is no apparent change because of
compensatory effect due to the decrease of microsomal yield
per g of tissue.
Pretreatment with MC, as with Aroclor, does not affect
the hepatic DMN demethylase in the mouse. Results not
tabulated show that, 24 hr following administration of MC,
40 mg/kg body weight, the control and experimental
enzyme activities (as nmoles HCHO per hr per mg micro
somal protein) were I55.97 ± I.05 and I50. 13 ±6.79,
oxidases (I , 6), is typical in its in vivo action on azo dye
N-demethylase and DMN demethylase in the rat, in that it
increases the activity of the former and decreases the
activity of the latter enzyme. Aroclor extends the spectrum
of agents that were known to induce hepatic azo dye
N-demethylase activity in the rat. MC, a variety of other
polynuclear hydrocarbons (4, 13), phenobarbital
(I 1), and
pregnenolone-l6a-carbonitrile
(J. C. Arcos, G. M. Bryant,
respectively (0.50 > p > 0.40). There is also no change in and M. F. Argus, unpublished observation) induce the en
enzyme activity when the MC dose is increased up to 160 zyme. Consistent with the induction of azo dye N-demeth
ylase by Aroclor in the rat, a metabolic step leading to a
mg/kg.
Effect of Aroclor 1254 on Hepatic Azo Dye N-Demethyl
ase. The results in Table 2 show that Aroclor induces this
considerable
enzyme system in both rats and mice; induction is notably
larger in the former species. The demethylase is also
induced in mice by MC pretreatment; the much larger
induction of this enzyme system by hydrocarbons in the rat
cinogenesis in the rat by 3'-methyl-4-dimethylaminoazoben
is well known (cf Refs. 4 and 13). Starvation has no
statistically demonstrable effect on azo dye N-demethylase
in the mouse.
presents a significant species-dependent
difference. While
the induction of liver tissue proliferation and of azo dye
N-demethylase
activity is maintained,
pretreatment
by
of aminoazo
dyes.
zene (23). Like other enzyme inducers, Aroclor also stimu
lates the synthesis of liver tissue in the rat.
The response of the mouse to Aroclor administration
Pretreatment with both MC and Aroclor also stimulates
the formation by the microsomes of polar dye metabolites
that do not migrate in thin-layer chromatography. As a
measure of the formation of these metabolites, the ratio of
the experimental over control absorbances was determined
using eluates of the nonmigrating spots. These ratios,
determined only in mice, were 7. 11 ±2.05 for Table 2,
Row 2;7.91±2.08forTable2,Row 3;4.19±1.22for
Induction ofhepatic
loss of carcinogenicity
PCB's have a substantial inhibitory effect on hepatocar
Aroclor following various administration schedules has no
effect on DMN demethylase. The response to MC of the 2
hepatic enzymes in the mouse is similar to their response to
Aroclor. The hydrocarbon is a moderately active inducer of
azo dye N-demethylase and has no effect on DMN demeth
ylase. However, MC brings about no change in the liver
weight/body weight ratio in mice, while in rats pretreatment
with aromatic hydrocarbons has been repeatedly shown to
Table 2
azo dye N-demethylase activity by Aroclor
/254 and MC in rats and mice
Sprague-Dawleymale rats (weight range,75 to 90 g) and Swiss-Webstermale mice (weight range,20 to 26 g) were used.
nmoles 3-methyl-4-aminoazobenzene
formed/hr/mg
postmitochondrialSpeciesAdministration
ExperimentalI.ratAroclor
schedule―proteinPercentSignificanceControl
0.001(5)52.mouseAroclor4 days prior to sacrifice35.08
±4. l2c
@
0.013.mouseAroclor
4 days prior to sacrifice (3)244.44
Iand 4 days prior to sacrifice169.32
@
(3)4.mouseAroclor 12hr starvation―
0.055.mouseMethylcholanthrene
24 hr prior to sacrifice (3)165.36
0.001sacrifice
87.60±3.60150p
±23.56
±4.08
24 hr prior to142.32
<
345.80 ±10.0441.50.02
295.08 ±14.8074.3p
±14.84
246.12±29.2048.80.10
±3.28
204.48 ±4.4044p
±9.96
186.52±14.081
> p >
0.00
p >
<
(3)6.mouseNo
0.20)(4)
administration, 24hr starvation210.60
> p >
1 (decrease)(0.30
aAroclor 1254or MC in corn oil were administeredby a single i.p. injection at the levelsof 500 and 40 mg/kg, respectively.
a Numbers inparentheses,
number of individual
determinations;
each determination
was carriedout induplicate.
C Mean
d
Both
±
S.D.
controls
and
experimentals
were
starved
for
the
last
12
hr
preceding
sacrifice
in
conformity
with
the
methodology
of
Czyganet al. (14).
JUNE 1975
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I577
M.
F.
Argus
et
a!.
produce substantial liver hypertrophy (4, 13, 16, 27, 39).
Starvation (by way of the release of carbohydrate repres
sion) produces substantial increase in the levels of various
mammalian enzymes (e.g., Refs. 26, 32 and 40) as well as
hepatic mixed-function oxidases in the rat, such as the azo
reductase (21) and DMN demethylase (34). The present
results indicate that starvation also brings about a small but
significant induction of DMN demethylase in the mouse.
Starvation does not influence, however, azo dye N-demeth
ylase activity in the mouse.
Using the same strain of animals, of the same sex and
age, originating from the same source, and approximating
their experimental conditions, we obtained results at van
ance with those reported by Czygan et a!. (14) that Aroclon
1254 is a powerful inducer of the demethylation of DMN in
the mouse liver. While we are at a loss to explain this
discrepancy, it may not be excluded that unknown environ
mental factors and seasonal and circadian influences may
have played a role. Nevertheless, the realization by these
authors of the multiplicity of cytochrome P-450-dependent
mixed-function
oxidases was not clear (see Ref. 28,
“Methods―).Yet, evidence for the multiplicity of micro
somal mixed-function oxidases is gathering from at least 2
directions. First, there is the realization of the repression of
certain mixed-function
oxidases by well-known inducers:
the repression of the DMN demethylase reported by us and
confirmed by Somogyi et a!. (29) is 1 instance of this
situation. Other examples are the decrease of N-demethyla
tion of mepenidine and Benadryl (12) and the inhibition of
hexobarbital
metabolism
(10) following benzo(a)pyrene
pretreatment.
Also, inhibition of zoxazolamine metabolism
following pretreatment of the animals with certain polycy
clic hydrocarbons
and their heterocyclic analogs has been
reported (e.g., Ref. 8). Second, chromatographic
and elec
trophoretic resolution of the microsomal hemoproteins (e.g.,
Refs. 9 and 38) provides direct evidence that a multiplicity
of P-450 cytochromes exist in liver microsomes.
7.
8.
9.
10.
II.
12.
Chlorine Containing Aroclors on Hepatic Mixed Function Oxidase.
Res. Commun. Chem. Pathol. Pharmacol., 3. 505-512, 1972.
Bresnick, E., and Stevenson, J. G. Microsomal N-Demethylase
Activity in Developing Rat Liver after Administration of 3-Methyl
cholanthrene. Biochem. Pharmacol.. 17: 1815- 1822, 1968.
Buu-Hoi. N. P., and Hien, D. P. Un Effet Biolo@ique Nouveau de
Certains Hydrocarbures Polycycliques Aromatiques et de Leurs
Analogues Hétêrocycliques:I'lnhibition
de l'Hydroxylation
de Ia
Zoxazolamine chez Ic Rat. Compt. Rend., 268: 423@426, 1969.
Comai, K., and Gaylor, J. 1. Existence and Separation of Three
Forms of Cytochrome P-450 from Rat Liver Microsomes. J. Biol.
Chem., 248:4947-4955,1973.
Conney, A. H., and Burns. J. J. Factors Influencing Drug Metabolism.
Advan. Pharmacol.. I. 31-58, 1962.
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I579
Effect of Polychlorinated Biphenyls (Aroclor 1254) on Inducible
and Repressible Microsomal N-Demethylases in the Mouse and
Rat
Mary F. Argus, Georgia M. Bryant, Karen M. Pastor, et al.
Cancer Res 1975;35:1574-1579.
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