United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
vvEPA
Research and Development
EPA-600/S1-81-010 Apr 1981
Project Summary
Effects of Halogenated
Aromatic Compounds on the
Metabolism of Foreign
Organic Compounds
Gary P. Carlson
This work was initiated to extend
our previous findings on the induction
of xenobiotic metabolism by the halo-
genated benzenes. Particular interest'
was focused on studying further the
relationship between their long-term
induction and their storage in body fat.
A second objective was to determine if
the brominated benzenes caused he-
.patic porphyria, similar to that observed
with the fungicide hexachlorobenzene.
A third aim was to extend our observa-
tions to other halogenated aromatic
compounds. The final objective was to
examine the role of the halogenated
benzenes in the enhancement of es-
teratic pathways of xenobiotic me-
tabolism. Sprague-Dawley rats were
used for all experiments involving
animals, with the exception of Swiss-
Webster mice (Laboratory Supply Co.,
Indianapolis, IN) being used to mea-
sure esterases and pesticide toxicity.
In contrast to hexachlorobenzene,
the brominated benzenes, including
the fully substituted hexabromoben-
zene, did not induce hepatic porphyria
to any significant degree and did not
cause increases in the excretion of
porphyrins.
A number of trichlorophenols were
assessed for their potential to increase
xenobiotic metabolism. They did not
increase any of the indicators of induc-
tion.
The brominated diphenyl esters.
commercial mixtures used as fire
retardants, were found to be potent
inducers of xenobiotic metabolism.
Administration of low doses for 90
days resulted in effects which were
prolonged far beyond the period of
administration. The chlorinated di-
phenyl ether isomers did not share this
potent induction ability.
The chlorinated benzo-p-quinones
examined were not found to be in-
ducers, but did demonstrate a very
strong cumulative toxicity.
Studies on the distribution and
elimination of 1,2,4-trichlorobenzene
and 1,2,4-tribromobenzene indicated
their prolonged inductive effects were
related to storage and slow release,
particularly in adipose tissue. Aroclor
1254 caused prolonged increases in
xenobiotic metabolism, which could
also be enhanced by starvation.
The halogenated benzenes induced
esterases associated with the metabo-
lism of acetanilide, procaineand phen-
yl acetate. They were able to protect
against the lethality and inhibition of
cholinesterase activity associated
with the organophosphate insecticides
parathion and malathion, plus their
active metabolites, paraoxon and
malaoxon. This protection was accom-
plished by enhancing the metabolic
detoxification of these compounds via
both exterases and mixed function
oxidases.
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This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Cincinnati. OH. to announce
key findings of the research project
which is fully documented in a separate
report of the same title (see Project
Report ordering information at back.)
Introduction
There is great interest in the induction
of xenobiotic metabolism by halogenated
aromatics, and the resultant interactions
of these compounds with the actions of-
concomitantly administered chemicals
or naturally occurring chemicals, such
as hormones. Most studies, however,
have focused on the polychlorinated
biphenyls, polybrominated biphenyls,
and chlorinated dibenzo-p-dioxins. Pre-
vious studies in our laboratory have
indicated that the chlorinated and
brominated benzenes are also inducers
of xenobiotic metabolism.
There are definite structure-activity
relationships, with brominated benzenes
more active than chlorinated analogs. In
general, the greater the degree of
halogenation, the greater the degree of
induction. Second, effects are prolonged
beyond the period of administration of
the halogenated benzenes.
Observations pertaining to the actions
of halogenated benzenes on mixed
function oxidases led to the necessity of
extending the investigation to; actions
on porphyria and esterase activity,
examining the relationship between
storage of halogenated benzenes and
their long-term effects, and exploring
the possibility that other halogenated
aromatic compounds might also be
inducers of xenobiotic metabolism.
Hepatic Porphyria
Hexachlorobenzene has been ob-
served in a number of laboratory species
and in humans to cause more porphy-
rinogenic effects than do the less
chlorinated benzenes. Brominated com-
pounds are often more active than
chlorinated analogs. Therefore it be-
came important to investigate the effects
of the fire retardant hexabromobenzene
(HBB) on porphyrin synthesis. In the
present studies, the effects of two less
halogenated benzenes, 1,4-dibromo-
benzene and 1,2,4-tribromobenzene,
were also examined.
Halogenated Aromatics as In-
ducers of Xenobiotic Metabo-
lism
Chlorinated benzenes induce micro-
somal enzymes associated with xeno-
biotic metabolism. Very little is known of
the potential of chlorinated phenols. It
appeared possible that the chlorinated
benzenes may not be direct inducers of
xenobiotic metabolism, but indirectly
with conversion to chlorinated phenols.
This study was undertaken to determine
whether or not trichlorophenols could
alter xenobiotic metabolism.
Brominated diphenyl ethers are struc-
turally similar to several halogenated
aromatic compounds known to induce
xenobiotic metabolism. These com-
pounds are commercial mixtures, making
it imperative to compare them with
diphenyl ether itself, bis(p-bromodi-
phenyl) ether, and the fully brominated
diphenyl ether.
Chlorinated diphenyl ethers are con-
taminants of chlorinated phenols and
readily concentrated, for example, by
fish. Because of the structural-activity
relationships among chlorinated bi-
phenyls and chlorinated dioxins, the
chlorinated diphenyl ethers were ex-
amined. Also, the potency of individual
isomers wer,e compared to commercial
mixtures of brominated diphenyl ethers.
Benzoquinones were examined for
effects as inducers. Although they did
not qualify in this respect, they were
found to have very cumulative toxicity.
Benzofurans were very potent inducers
of xenobiotic metabolism.
Relationship between Induction
and Storage
Municipal wastewaters contain sev-
eral chlorinated and brominated ben-
zenes which induce xenobiotic metabo-
lism at low doses. Brominated isomers
are generally more potent inducers of
hepatic microsomal enzymes than chlo-
rinated isomers, and the 1,2,4-trihalo-
genated compound is a more potent
inducer than other isomers.
Studies with 1,2,4-trichlorobenzene
and 1,2,4-tribromobenzene show these
compounds cause enzyme induction,
even at low doses. Observations sug-
gest storage in body tissues, with slow
release after dose termination, thereby
prolonging induction of xenobiotic me-
tabolism.
Numerous studies have shown poly-
chlorinated biphenyls induce xenobiotic
metabolism. In view of the results with
the halogenated benzenes, similar
experiments were conducted to deter-
mine the length of time PCBs affect
xenobiotic metabolism.
Esterase Activity
Little is known about the ability of
halogenated and chlorinated benzenes
to alter esterases, except that over 200
pharmacologically active compounds
are esters. Therefore hexabromoben-
zene was compared to less halogenated
benzenes regarding the metabolism of
acetanilide, which is biotransformed by
both an oxidative reaction and an ester-
ase. Hydrolysis of procaine was also
studied.
Conclusions
This study increased in scope and
depth as research progressed. The
interactions affecting xenobiotic me-
tabolism were more complex than origi-
nally hypothesized. The more obvious
conclusions are stated under subhead-
ings; hepatic prophyria, halogenated
aromatics, relationship between indue-
tion and storage, and esterase activity.
Hepatic Porphyria
The brominated benzenes did not
significantly increase hepatic porphyria.
The chlorinated benzenes, other than
hexachlorobenzene, had previously
been shown to cause minimal increases
in liver or urinary porphyrins. Similarly,
hexabromobenzene, 1,4-dibromoben-
zene, and 1,2,4-tribromobenzene (TBB)
caused only small increases in hepatic
porphyrins, but did not cause increases
in ALA synthetase or in the urinary
excretion of porphobilinogen, amino-
levulinic acid, or porphyrins.
Halogenated Aromatics
The trichlorophenols, which in some
species are metabolites or contami-
nants in halogenated benzenes, were
not inducers of xenobiotic metabolism.
The brominated diphenyl ethers were
potent short-term inducers of xenobi-
otic metabolism, as well as potent long-
term (90 days) inducers at low doses.
Measurements of these ethers in tissue
were not made, but it is likely that the.
extended period of induction, observable
30 to 60 days after the last dose, was a
result of their induction potential and i
accumulation in sites such as adipose
tissue and liver.
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Chlorinated diphenyl esters were
inferior as inducers of xenobiotic meta-
bolism compared to what would be
predicted from studies on commercial
brominated diphenyl ether mixtures.
Diphenyl ether isomers with 2 or 3
chlorines were inactive at the experi-
mental doses. However, similar PCBs
exhibited low-induction potential. Pen-
tabromodiphenyl and octabromodiphen-
yl ether mixtures were more active than
4,4'-dibromodiphenyl ether. The more
chlorinated the diphenyl ethers, the
better their induction. Curiously, deca-
chlorodiphenyl ether treatment caused
a shift in the P-450 peak toward 448,
but did not induce AHH; although 2,4,5,
3',4-pentachlorodiphenyl ether caused
both effects. The degree of substitution
and position of these groups were both
important to determine the extent and
type of induction.
The toxicity of benzoquinones was
very cumulative. A dose far below that
of acute toxicity was lethal when given
daily for 17 to 30 days. The reasons for
this toxicity were not readily apparent.
One possibility considered and discarded
as erroneous was the occurrence of
methemoglobin formation. Both of the
chlorinated dibenzofurans induced
benzpyrene hydroxylase activity, but
dibenzofuran itself did not.
Relationship between Induc-
tion and Storage
Both TBB and TCB induced hepatic
microsomal enzymes for at least 16
days, after ending 7 days of a 1 mmolper
kilogram dose per day. Excretion data
indicated more TBB than TCB was held
in the body. Metabolism of trihalogen-
ated benzenes to trihalogenated phe-
nols has been demonstrated in rabbits,
but these were not inducers of xeno-
biotic metabolism in rats. The major
metabolites in rats appeared to be
mercapturic acid derivatives.
Halogenated benzenes were observed
in tissues. Increased induction resulting
from 4 days of starvation, at 6 days after
TBB administration was terminated,
suggested TBB was mobilized from fat
depots. Phenobarbital treatment imme-
diately after halogenated benzene dosing
appeared to hasten mobilization, meta-
bolism, and excretion of either com-
pound. This left smaller amounts in the
rat to cause" induction at the time of
assay.
It was clear that both TBB and TCB
were retained in the body and released
slowly after administration. On an equi-
molar basis, TBB led to higher levels of
hepatic enzyme induction. These levels
were maintained for longer periods and
retained to a greater extent in body
tissues. TBB was influenced more by
starvation than TCB. Starvation accel-
erated excretion of halogenated benzene.
For phenobarbital treatment, this effect
was inferred from the decrease in
enzyme levels, compared to those in
non-phenobarbital-induced animals. A
similar relationship of time and loss of
elevated xenobiotic metabolism was
seen for polychlorinated biphenyls. This
induction was also increased by starva-
tion.
Administration of the commercial
polychlorinated biphenyl mixture, Aroclor
1254, resulted in increases in xeno-
biotic metabolism. This persisted for as
long as 4 to 5 months.
Esterase Activity
Halogenated benzenes induced xeno-
biotic metabolism by increasing ester-
ase activity. This applied to phenyl
acetate, acetanilide, and procaine in-
dicating broad induction of oxidative,
reductive, conjugative, and esteratic
metabolic reactions. The compounds
most active in inducing arylesterase
activity in the liver were 1,2,4-TCB and
1,2,4-TBB, shown throughout our
studies as better inducing isomers.
Trihalogenated benzenes decreased
the inhibitory effect of malathion on
cholinesterase activity in the brain, and
to a lesser degree in red blood cells, but
not in liver or plasma. It therefore
appeared that halogenated benzenes
protect against malathion or malaoxon
toxicity by increasing the detoxification
rate.
The trihalogenated benzenes de-
creased the inhibitory effect of paraoxon
on cholinesterase activity in the brain
and liver, but not in plasma or red blood
cells. Thus, it appears that the halo-
genated benzenes protect against para-
oxon and probably parathion toxicity by
increasing the detoxification rate of the
pesticides.
Results
Basically, xenobiotic metabolism was
induced by chlorinated and brominated
benzenes. This induction affects both
mixed function oxidases and esterases.
Porphyria was not caused by chlori-
nated benzenes, except for hexachloro-
benzene.
Administration of 1,2,4-tribromoben-
zene caused a significant increase in
liver weight, even at the lowest level of
50 mg/kg in 30 days. An increase m
porphyrin content in the liver did not
appear until 90 days (Table 1). The
Table 1.
Dose
(mg/kg)
0
50
100
200
0
50
100
200
0
50
100
200
0
50
100
200
Effect of 1,2,4-Tribromobenzene PO on Porphyrin Production and Excre-
tion in Female Rats
Liver
porphyrins Urine porphyrins
Liver wt (g) (ng/g) (ng/24 h)
6.46 ± 0.34s
8.94 ±0.56"
8.94 ± 0.56"
9.52 ±0.30°
7.30±0.21a
9.16 ±0.42"
9.96±0.37ti
9.72 ±0.50b
6.44 ± 0.26s
8.97 ± 0.43"
10.07 ± 0. 18*
12.20±0.88C
7.27 ± 0.38*
11.11 ±0.80b
12.71 ±0.84*
15.18 ±0.23C
30 days of administration
526 ± 49*
602 ± 90*
620 ± 66a
471 ±22*
60 days of administration
156 ± 64"
251 ± 75a
285 ± 78a
2.85 ±54*
90 days of administration
468 ± 12*
629 ± 2d°
700 + 17C
711 ±26C
120 days of administration
411 ± 13*
560 ± 33b
646 ± 28b'c
682 ± 45C
2.1 9 ±0.28*
2.29 ±0.69*
2.40 ±0.13*
2.53 ± 0.27*
1.92 ±0.58*
1.61 ±0.21*
1.77 ±0.48*
2.42 ± 0.54*
1.41 ±0.23*'*
1.17 ±0.18*
2.43+0.59"
1 .88 ± 0.35*'"
1.75 ±0.39*
1.81 ±0.42*
2.27 ± 0.56*
2. 13 ± 0.40*
cVa/ues with same superscript are not significantly different fp>0.05J.
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increases were dose dependent, small
and very similar to those previously
observed with 1,2,4-trichlorobenzene.
Animals treated with 200 mg/kg of
1,2,4-tribromobenzene for 120 days
were slightly smaller than the controls,
but not statistically significant. They
were discolored and their ears brown
and ragged in appearance. Thus, al-
though the animals looked unhealthy,
porphyria was not the problem.
Hexabromobenzene was not shown
to share the extreme porphyrinogenic
properties of its chlorinated analog. The
less brominated compounds were simi-
lar to the chlorinated analogs in being
very weak porphyria inducers.
Table 2. Effect of Trichlorophenols In Vitro on p-Nitroanisole O-demethylation and
EPN Detoxification
p-Nitroanisole
0-demethylation0 EPN detoxification0
(fjg p-nitrophenol/ (t*g p-nitrophenol/
Treatment 50 mg/30 min) 50 mg/30 min)
Control"
2,3,5-Trichlorophenof1
2,3,6- TrichlorophenoP
2,4,5- TrichlorophenoP
2, 4, 6- TrichlorophenoP
6. 64 ±0.68
0.57 ± 0.21"
2.02 ±0.22"
0.61 ±0.24"
2.33 ± 0. 19"
8.39 ± 0.34
4. 02 ± 0.43d
4.58 ± 1.14"
3.88 ± 0.47"
4.03 ± 1.1 '7"
*50 /j( benzene added
^Compound added in 50 /jl benzene to give final concentration of 2.5 x 10'* M.
cMean ± S.E. of 4 experiments.
^Significantly different from control (p<0.05).
Halogenated Aromatics
Chlorinated Phenols—
All of the experimentation resulted in
an excess of negative data for halogena-
ted aromatic induction of xenobiotic
metabolism. These compounds did not
induce EPN detoxification, NADPH
cytochrome c reductase, or cytochrome
P-450. Specific compounds, such as
1,2,4-trichlorobenzene and 1,3,5-tri-
chlorobenzene, did not depend upon
biotransformation to trichlorophenols
for induction ability, so they must be
regarded as inactive metabolites.
In vitro the trichlorophenols were
inhibitors of EPN detoxification and p-
nitroanisole demethylation (Table 2).
For p-nitroanisole demethylation, the
inhibition was noncompetitive. The 2,3,5-
and 2,4,5-isomers were the most potent.
Brominated Diphenyl Ethers—
The brominated diphenyl ethers,
including the complex commercial mix-
tures of pentabromodiphenyl and octa-
bromodiphenyl ethers, (Table 3), caused
liver enlargement (Table 4). Pentabro-
modiphenyl ether gave the largest
increase of 64%. A similar pattern was
seen with both NADPH cytochrome c
reductase and cytochrome P-450 using
pentabromodiphenyl ether.The effect of
pentabromodiphenyl ether was greater
than that of octabromodiphenyl ether,
which was equal to bis(p-bromodiphenyl)
ether. Decabromodiphenyl ether was
without effect. This was similar to the
poor induction properties of fully bromi-
nated benzene.
When a series of enzyme activities
were determined, pentabromodiphenyl
ether and octobromodiphenyl ether
Table 3. Composition of Bromodiphenyl Ethers
Mol wt
Percent of bromodiphenyl
Compound 02 4
Diphenyl ether 98
Bis (p-bromophenyl) ether *
Pentabromodiphenyl ether 24. 6
Octabromodiphenyl ether
Decabromodiphenyl ether
5 6
58.1 13.3
1.1 8.5
7 8
2.6 0.3
45.1 30.7
con-
glom-
erate
9 10 M"' ""
170
328
0.2 0.8 564
13.0 1.6 766
" 959
"Reagent grade.
"High purity.
Table 4. Effect of Bromodiphenyl Ethers on Liver wt./Body wt., NADPH Cyto-
chrome c Reductase and Cytochrome P-450
Cytochrome c
reductase, nmol
Treatment"
Control
Diphenyl ether
Bis (p-bromophenyl) ether
Pentabromodiphenyl ether
Octabromodiphenyl ether
Decabromodiphenyl ether
Liver wt. inn"
Body wt.
3.80 ± 0.1 2C
4.03 ± 0. 13C'"
4.48 ±0.1 2**
6.25 ± 0.25'
5.54 ± 0.259
4.75 ±0.10'
Cyto. c reduced/
min/mg protein*
151 ± 26C
134 ± 25°
238 ± /3d*
315 ±20'
281 ± 34'''
175± 5C"
Cytochrome P-450
nmol/mg protein*
0.79 ± O.O5C
0.71 ±0.04°
1.61 ± 0.09"
2.56 ±0.1 7*
1.86 ±0.14"
0.91 ± 0.05°
'0.1 mmol/kg/day for 14 days po. Controls received corn oil.
tiMean ± S.E. for 4 rats.
°~a Values with the same superscript are not significantly different from one another
(p >0.05).
were the more potent inducers, giving
large increases in both EPN detoxifica-
tion and p-nitroanisole demethylation
(Table 5). They were the only compounds
which caused increased conjugation of
naphthol. Benzo(a)pyrene hydroxylase
was increased only by pentabromo-
diphenyl ether.
Administration of 6.25, 12.5 or 25
/ymol/kg daily of the pentabromo-
diphenyl ether and octabromodiphenyl
ether over a 90-day period resulted in
large increases in EPN detoxification, p-
nitroanisole demethylation, and cyto-
chrome P-450 (Table 6). Pentabromo-
diphenyl ether did not cause large
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Table 5. Effect of BromodiphenylEthers on EPNDetoxification, p-Nitroanisole Demethylation, Benzo(a)pyreneHydroxylase, and
UDP-Glucuronyltransferase
p-Nitroanisole UDP-Glucuronyl-
Treatment*
EPN detoxification,
fjg p-nitrophenol/
50 mg/30 min°
demethylation,
jjg p-nitrophenol/
50 mg/30 min°
transferase,
nmol napthol/
mg protein/min*
Benzola)pyrene
hydroxylase, nmol/mg
protein/10 min*
Control
Diphenyl ether
Bis (p-bromophenyl) ether
Pentabromodiphenyl ether
Octabromodiphenyl ether
Decabromodiphenyl ether
6.60 ± 1.1 2C
11.08± 1.72"
10.42± 1.24c'a
15.98 ± 1.22*
16.30 ± i.ir
7.10 ±0.85°
6.33 ±0.7 8^
5.68 ± 0.85°
9.25±0.75a
43.81 ± 1.93e
17.84 ±0.41'
8.68 ± 1.06c'a
6.23 ± 1.25C
5.76±0.73C
7.65 ± 1.12c'a
10.87 ± 1.74a
10.36 ± 1.14a
5.97 ±0.98C
6.79±0.54C
4.44±0.55C
4.86±0.74C
11.01 ± 1.07a
6.01 ± 1.06C
5.52 ± 0.61C
aO. 1 mmol/kg/day for 14 days po. Controls received corn oil.
*Mean ± S.E. of 4 rats except: 3 rats in pentabromodiphenyl ether group in liver wt./bodywt. and glucuronyltransferase and 2 for
that group in benzo(a)pyrene hydroxylase.
c~'Values with the same superscript are not significantly different from one another (p >0.05).
Table 6. Effect of Pentabromodiphenyl Ether and Octabromodiphenyl Ether on EPN Detoxification, p-Nitroanisole Demethylation,
Cytochrome c Reductase Activity, and Cytochrome P-450 Content
p-Nitroanisole Cytochrome c
Dose EPN detoxification, demethylation, reductase, nmol
fjmol/kg/day tig p-nitrophenol/ fjg p-nitrophenol/ Cyto. c reduced/ Cytochrome P-450
for 90 days 50 mg/30 min 50 mg/30 min min/mg protein nmol/mg protein
0 9.6±0.3a
6.25 17.7 ±0.5*
12.5 2/.5±0.6c
25 26.5 ±0.7*
0 9.7±0.5a
6.25 21.3 ±1.1"
12.5 26.9 ±2.2*
25 37.1 ±2.0C
Pentabromodiphenyl ether
7.6 ±0.1a
25.6 ± 1.2*
34.6 ± 1.1C
50.1 ± 1.3d
Octabromodiphenyl ether
10.0 ± 0.4a
20.9 ± 1.8*
27.3 ± J.3C
38.4 ± 1.9*
88 ± 6*
117± 9*
120 ± 5b
138 ± 7*
171 ± 11a
197 ± 9a
196 ± 18a
208 ± 11a
0.60 ± 0.04a
1.02 ±0.06*
1.1 6 ±0.07*
1.1 4 ±0.07*
1.04 ±0.10"
1.56 ± 0. 13b
1.27 ± 0. 16at>
1.46 ±0.09*
Values with same superscript are not significantly different from one another (p > 0.05).
increases in NADPH Cytochrome c re-
ductase, nor did Octabromodiphenyl
ether even at the highest dose level.
When administered for 90 days, even
the lowest dose of pentabromodiphenyl
ether (0.78 /umol/kg daily) caused in-
creases in EPN detoxification, p-nitro-
anisole demethylation, NADPH cyto-
chrome c reductase, and cytochrome P-
450 (Table 7). For EPN detoxification
andp-nitroanisole demethylation, there
was a clear-cut dose response relation-
ship.
Chlorinated Diphenyl Ethers—
Chlorinated isomers proved to be poor
inducers of xenobiotic metabolism
when compared to the very potent
induction ability of the polybrominated
diphenyl ethers. Only two of the isomers
caused increases in aryl hydrocarbon
hydroxylase, 3,4,2,4-tetrachlorodiphenyl
ether and 2,4,5,3',4'-pentachloro-
diphenyl ether.
When microsomal NADPH cytochrome
c reductase activity was measured, only
decachlorodiphenyl ether caused an
increase. This compound caused the
largest increase in cytochrome P-450
content. Small but significant eleva-
tions were also observed in rats treated
with both 2,4,5,2',4',- and 2,4,5,3',4'-
pentachlorodiphenyl ether.
Dibenzofurans—
Dibenzof uran and its chlorinated deri-
vatives did not increase EPN detoxifica-
tion or NADPH cytochrome c reductase.
Benzypyrene hydroxylase activity was
induced by both chlorinated dibenzo-
furans, but not by the parent compound.
Both chlorinated dibenzofurans increased
cytochrome P-450 content, although
the increase was not statistically signifi-
cant with the 2,8-dichloro isomer. Both
also caused a shift to 448.2 nm for the
peak of cytochrome P-450 absorbance.
These factors strongly support the
contention that the compounds are
inducers of the 3-methylcholanthrene
type.
Relationship between Storage
and Induction
Halogenated Benzenes—
Due to the large difference in molecu-
lar weight of the halogenated benzenes
tested previously they were compared
on a molar basis. A dose of 1 mmol/kg/
day for 7 days was used in all studies.
This dose caused a high level of induction
on day 1 after the 7 days with both TBB
and TCB (Figures 1 and 2). EPN detoxifi-
cation levels were approximately twice
as high as control values 16 days after
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the last dose of TBB, suggesting induc-
tion may have occurred beyond day 16
of recovery. The decline in induction
was slower with TBB than TCB.
Duration of Induction by PCB
and Influence of Starvation—
The results of Aroclor 1254 adminis-
tration were as expected. EPN detoxifi-
cation andp-nitroanisole demethylation
were elevated four- and ten-fold, respec-
tively, 1 day after the last (14th) dose.
Measurements of microsomal NADPH
cytochrome c reductase and cytochrome
P-450 revealed similar results. Cyto-
chrome P-450 increases were observed
for the first 71 days, due to Aroclor
alone, before returning to control levels
at later time periods. Changes in NADPH
cytochrome c reductase, although not
as large, followed a similar time course.
Esterase Activity
Acetanilide and Procaine
Esterase—
Halogenated benzenes induced the
metabolism of acetanilide and procaine
by increasing esterase activity. This was
true of the less halogenated benzenes,
as well as hexachlorobenzene. These
compounds were broad inducers of
oxidative, reductive, conjugative, and
esteratic metabolic reactions.
Esterases and Pesticide Toxicity
The 1,4-dihalogenated benzenes and
hexabromobenzene had little effect on
the lethality of malathion. Both trihalo-
genated benzenes increased the LD50
values, which indicated a decrease in
lethality. The trihalogenated benzenes
also decreased the toxicity of the active
metabolite of malathion, malaoxon,
approximately two-fold. Even greater
protection against lethality was demon-
strated with parathion.
The effect of the trihalogenated ben-
zenes on cholinesterase activity in the
brain, liver, plasma, and red blood cells
was measured, since toxicity of the
organophosphates is related to inhibition
of cholinesterase. Malathion caused a
decrease in cholinesterase activity in
the brains of control mice, but not in
those treated with 1,2,4-trichlorobenzene.
The decreases were similar between
control and treated groups in other
tissues. Similarly, 1,2,4-tribromobenzene
treatment prevented the decrease in
brain cholinesterase following malathion
treatment, and slightly decreased the
malathion effect on red blood cells.
Measurement of carboxylesterase
activity with malathion as the substrate
revealed that 1,4-dichlorobenzene,
1,2,4-trichlorobenzene, hexachloroben-
zene, 1,4»dibromobenzene, 1,2,4-tri-
bromobenzene, and hexabromobenzene
all increased carboxylesterase activity
in the liver. A similar trend was seen in
plasma. The trihalogenated benzenes
caused the largest increases in activity.
Paraoxon dearylation was examined
since halogenated benzenes might pro-
tect against paraoxon toxicity by increas-
ing its metabolism. Plasma esterase
was not altered by 1,4-dihalogenated or
hexahalogenated isomers. Trihalogenated
isomers actually decreased this activity.
As expected, there was little activity in
either control or treated animals in the
105,000 x g liver supernatant. Microso-
mal esterase activity was increased by
1,2,4-trichlorobenzene, hexachloroben-
zene, and hexabromobenzene, but was
inhibited by 1,4-dibromobenzene and
1,2,4-tribromobenzene. However, the
mixed function oxidase dearylation in
liver microsomes was increased eightfold
by 1,2,4-tribromobenzene and to a
lesser extent by 1,4-dichlorobenzene,
hexachlorobenzene and 1,2,4-trichloro-
benzene.
Arylesterase—
None of the halogenated benzenes
tested caused any increase in serum
arylesterase activity. These compounds
included 1,2,4-trichlorobenzene, 1,3,5-
trichlorobenzene, hexachlorobenzene,
1,2,4-tribromobenzene, 1,3,5-tribromo-
benzene, and hexabromobenzene. In-
creased activity was observed in the
liver after 1,2,4-trichlorobenzene, hexa-
chlorobenzene, 1,2,4-tribromobenzene,
and 1,3,5-tribromobenzene. The com-
pounds, therefore, associated through-
out this research project as the better
Table 7. Effect of Pentabromodiphenyl Ether on EPN Detoxification, p-Nitroanisole Demethylation, NADPH Cytochrome c
Reductase, and Cytochrome P-450
p-Nitroanisole NADPH Cytochrome
Dose EPN detoxification, demethylation, c reductase, nmol
fjmol/kg/day i*g p-nitrophenol /jg p-nitrophenol Cyto. c reduced
for 90 days 50 mg/30 min 50 mg/30 min min/mg protein
Cytochrome P-450,
nmol/mg protein
0
0.78
1.56
3.13
0
0.78
1.56
3.13
0
0.78
1.56
3.13
A dministration
5.8 ±0.1* 5.5 ±0.4* 165 ±10*
8.1 ±0.2* 9.7 ±0.6" 215 ±13*
8.9 ±0.3* 72.2 ±0.8° 194 ± 27a'b
11.4±0.6C 16.9 ±0.4" 209 ±14*'*
Administration plus a 30-day recovery period
6.0 ±0.2* 5.5 ±0.4* 128 ± 9*
7.2 ± 0.2* 7.5 ± 0.3* 138 ± 7a'b
7.4 ± 0.5* 7.9 ± 0.4* 138 ± 8a'b
9.1 ±0.2C 10.7±0.9C 152 ± 6b
Administration plus a 60-day recovery period
6.3 ± 0.3* 3.6 ± 0.3* 155 ± 9*
6.4 ± 0.3* 3.8 ± 0.2* 162 ± 14*
7.7 ±0.3* 4.6 ±1.0* 148± 7*
7.5 ±0.4* 5.6 ±0.3° 772± 8"
7.05 ± 0.02*
1.41 ±0.07*
1.26 ±0.06*
1.25 ±0.09*
0.80 ± 0.07*
0.87 ± 0.04*
0.96 ± 0. 18*
0.90 ± 0. 13*
0.82 ± 0.07*
0.85 ±0.1 2a
0.85 ± 0.06*
0.82 ± 0.06*
a-d
For each time period, values with same superscript are not significantly different from one another (p > 0.05).
-------
500
400
o 300
§
200
;oo
0
*
*
fS
Days after—
Last Dose
6 11 16
EPN
Detoxification
1 6 11 16
p-Nitroaniso/e
Demethylation
6 11 1t
Cytochrome c
Reductase
6 11 16
Cytochrome
P-450
Figure 1. Decline with time in rats receiving TBB(1 mmol/kg/day for 7 days po. Asterisk indicates values significantly different
from controls IP 0.05). Average control values: EPN detoxification, 5.9 ±0.2 (jg p-nitrophenol/50 mg/30min;
p-nitroanisole demethylation, 5.1 + 0.3 (jgp-nitrophenol/SOmg/30 min; NADPH-cytochrome c reductase, 118±6
nmol cytochrome c reduced/min/ml protein; P-450. 0.85 + 0.07 nmol/mg protein.
inducing isomers, were also the most
active in inducing arylesterase activity
in the liver.
Recommendations
The results of these studies support
our earlier findings that many of the
halogenated aromatic compounds are
capable of altering the metabolism of
foreign organic compounds. These
reactions'include the mixed function
oxidases, conjugative enzymes, and
esterases. A critical area in need of
additional research is the relationship of
storage to prolonged induction.
In our studies on halogenated ben-
zenes and PCBs, the period of increased
xenobiotic metabolism lasted far beyond
the period of administration of the com-
pounds. The halogenated benzenes
were able to correlate this activity with
storage, especially in adipose tissue.
This is also suggested, but not proven,
for PCBs. More studies on PCBs and
brominated diphenyl ethers*are needed.
The chlorinated benzofurans were
found to be inducers, supporting pre-
vious findings. The chlorinated benzo-
quinones did not prove to be inducers,
but did show highly cumulative toxicity.
The reasons for this are not known, but
are evidently not related to methemoglo-
bin formation or liver damage. The
dramatic nature of this cumulative
toxicity indicates subsequent study is
needed to ascertain the mechanism
involved.
The brominated diphenyl esters were
among the most potent inducers tested
in our laboratory. Little is known about
their pharmacokinetic properties or
effects, other than those associated
with xenobiotic metabolism. Additional
studies are needed to quantify the
tissue distribution, metabolism, and
rate of elimination. These complex
mixtures of isomers need to be identified
to ascertain the active ones causing the
induction process.
-------
500
400
300
§
0)
0. 200
700
fc
i
A
Days after—
Last Dose
1 6 11 16
EPN
Detoxification
1
11 16
p-Nitroaniso/e
Demethylation
1 6 11 16
Cytochrome c
Reductase
6 11 16
Cytochrome
P-450
Figure 2. Decline with time in rats receiving TCB. Average control values: EPN detoxification, 6.3 ±0.4 fjg/50 mg/30 min;
p-nitroanisole demethylation. 5.1 ± 0.2/jg/50 mg/30 min; NADPH-cytochrome c reductase, 118 + 6 nmol/min/mg;
P-450, 0.85 + 0.07 nmol/mg.
Gary P. Carlson is with the Department of Pharmacology and Toxicology, Purdue
University, West Lafayette, IN 47907.
Merrel Robinson is the EPA Project Officer (see below}.
The complete report, entitled "Effects of Halogenated Aromatic Compounds on
the Metabolism of Foreign Organic Compounds," (Order No. PB81 -152 522;
Cost: $9.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
i US GOVERNMENT PRINTING OFFICE 1981-757-064/031Z
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