TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
Metabolism
103,102- 112 (1990)
of the Atylamide
Herbicide Propanil
II. Effects of Propanil and Its Derivatives on Hepatic Microsomal
Drug-Metabolizing Enzymes in the Rat’
DAVID C. McMILLAN,*,~-*
JOELLYN
*National
Centerfor
Interdisciplinary
Toxicological
Toxicology,
JULIAN E. A. LBAIu%,*,t
MICHAEL P. ARLOTTO,*
M. MCMILLAN,*‘*
AND JACK A. HINSON*,?~
Research, Jefferson, Arkansas
72079, and the TDepartment
ofPharmacology
University
of Arkansas for Medical Sciences, Little Rock, Arkansas
72205
Received
July 18. 1989; accepted
October
and
26, I989
Metabolism of the Arylamide Herbicide Propanil. II. Effects of Propanil and Its Derivatives
on Hepatic Microsomal Drug-Metabolizing
Enzymes in the Rat. MCMILLAN,
D. C., LEAKEY,
J. E. A., ARLOTTO,
M. P., MCMILLAN,
J. M., AND HINSON, J. A. ( 1990). Toxicol. Appl. Pharmacol. 103, 102- 112. Propanil(3,4-dichloropropionanilide)
is an arylamide herbicide that has been
reported to be contaminated with the cytochrome P450 enzyme inducers 3,3’,4,4’tetrachloroazobenzene (TCAB) and 3,3’,4,4’-tetrachloroazoxybenzene
(TCAOB), which are
structural analogs of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). We determined if treatment
of rats with TCAB, TCAOB, propanil, 3,4dichloroaniline,
TCDD, or phenobarbital induced
the hepatic microsomal metabolism of propanil and 3,4dichloroaniline. Acylamidase-catalyzed
hydrolysis of propanil to 3,4dichloroaniline
was not induced by any of the pretreatments; however, hydroxylation of propanil at the 2’position was induced by TCDD, TCAB, TCAOB, propanil, and 3,4dichloroaniline pretreatments. Ring- and N-hydroxylations of 3,4dichloroaniline
were induced by TCDD, TCAB, TCAOB, and 3,4dichloroaniline
pretreatments. Microsomal
7-ethoxyresorufin-Odeethylase (EROD) and 7-benzoxyresorufin-Odealkylase (BROD) activities and electrophoretic mobility of microsomal proteins suggested that cytochromes P45Oc and
P450d were induced by TCAB and TCAOB pretreatment. EROD, BROD, and 7-pentoxyresorufin-Odealkylase activities were slightly increased in microsomes from propanil- and 3,4dichloroaniline-pretreated
rats, which suggests that these compounds may be weak inducers of
cytochrome P450 isozymes. 0 1990 Academic Press, Inc.
Propanil (3,4-dichloropropionanilide)
is one
of the most extensively used herbicides in
rice-growing regions of the United States
(Bartha and Pramer, 1970; Brewster, personal communication,
1985). Propanil has
been reported to be contaminated
with
3,3’,4,4’-tetrachloroazobenzene
(TCAB) and
’ This work was presented at the Society of Toxicology, Atlanta, GA, 1989 [Toxicologist
9, 195 ( 1989)].
Z Present address: Department of Pharmacology, Medical University of South Carolina, Charleston, SC 29429.
3 To whom correspondence should be addressed:
HIT-1 10, National Center for Toxicological Research,
Jefferson, Arkansas 72079.
0041008X/90
$3.00
Copyright 0 1990 by Academic Rs, Inc.
All tights of reptuduction
in any form reserved.
3,3’,4,4’-tetrachloroazoxybenzene
(TCAOB)
(Hill et al., 1981; Di Muccio et al., 1984),
which are structural analogs of the well-characterized drug-metabolizing
enzyme inducer
2,3,7&tetrachlorodibenzoqdioxin
(TCDD)
(Fig. 1). The toxicities and Ah-receptor binding characteristics of TCAB and TCAOB are
similar to those of the more potent TCDD
(Poland and Knutson, 1982). Furthermore,
occupational exposure to these contaminants
in technical-grade propanil has resulted in
several occurrences of chloracne (Morse et
al., 1979; Kimbrough,
1980). Although
TCAB and TCAOB have been demonstrated
to be Ah-dependent inducers of the cyto102
ENZYME
INDUCTION
BY PROPANIL
DERIVATIVES
chrome P450 system (Poland et al., 1976;
Hsia and Kreamer, 1979), their effects on specific isozymes of cytochrome P450 and other
Ah responsive enzymes, such as UDPglucuronyltransferase
(Owens, 1977), have
‘Cl
not been investigated. In addition, the possi3,3’,4,4’-Tetrachloroazoxybenzene
bility that propanil or 3,4dichloroaniline
(TCAOB)
may also induce cytochrome P450 has not
been systematically examined.
The acute toxicity of propanil is manifested primarily by methemoglobin
formation, which has been reported to lead to cyanosis in occupationally
exposed humans
(Kimbrough,
1980). Propanil is thought to
Cl
produce methemoglobinemia
following a se3,3’,4,4’-Tetrachloroazobenzene
ries of enzymatic reactions beginning with
(TCAB)
acylamidase-catalyzed
hydrolysis to 3,4-dichloroaniline, followed by cytochrome P450catalyzed oxidation of 3,4dichloroaniline
to
toxic metabolites (Singleton and Murphy,
1973). In the previous report (McMillan
et
al., 1990) the major pathways of propanil
and 3,4dichloroaniline
metabolism
in rat
2,3,7,8-Tetrachlorodibenzo-pdioxin
liver microsomes were identified. In addition,
(TCDD)
we showed that the metabolites
N-hyFIG. 1. Structures of 3,3’.4.4’-tetrachloroazobenzene
droxy-3,4-dichloroaniline
and 6-hydroxy(TCAB). 3,3’,4,4’-tetrachloroazoxybenzene
(TCAOB).
3,4-dichloroaniline
may mediate propaniland 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD).
induced methemoglobinemia
because these
derivatives were able to directly oxidize
hemoglobin in vitro (McMillan et al., 1990).
In this study we have examined the effects mCi/mmol) was purchased from Chemsyn Science Laboratories (Lenexa, KS). Propanil and [ring-U-14C]propaof induction with TCDD and phenobarbital
on the metabolism
of propanil and 3,4- nil ( I. 13 mCi/mmol) were synthesized from nonlabeled
and radiolabeled 3,4-dichloroaniline, respectively, as dedichloroaniline
in rat liver microsomes (Fig. scribed
previously (Lay et al.. 1986). These compounds
2). In addition, we compared the effects of were determined to be greater than 99% pure by highTCAB, TCAOB, propanil, and 3,4dichloroperformance liquid chromatography. 2.3,7,8-Tetraaniline pretreatments on propanil and 3,4- chlorodibenzo-p-dioxin was a generous gift of Dr. Stedichloroaniline
metabolism as well as the in- phen Safe, Texas A&M University,.College Station.
duction of several hepatic microsomal drug- Texas. TCAOB and TCAB were synthesized as described
by Hsia and Burant (1979). Ethoxy- and pentoxyresorumetabolizing enzymes.
fin were synthesized from resorufin (Matherson, ColeMATERIALS
AND
METHODS
Chemicals
Pyridine nucleotides were purchased from Sigma
Chemical Company (St. Louis, MO). 3,4-Dichloroaniline was purchased from Aldrich Chemical Company
(Milwaukee, WI). 3,4-[ring-U-“‘C]Dichloroaniline
(1.24
man, and Bell, Norwood, OH) and the appropriate iodoalkanes (Aldrich) by the method of Prough et al.
(1978). Benzoxyresorufin was purchased from Molecular
Probes (Eugene, OR). Purified rat hepatic cytochromes P450a (P450IIA1),4 P450b (P45&Bl),
P45Oc
(P45OIAl). P45Od (P450IA2). P45Og (P45OIIC13). and
4 New cytochrome P450 nomenclature as presented by
Nebert and Gonzalez ( 1987).
MCMILLAN
104
ET AL.
I:
H-N-C-CH2-CH3
P’-OH-Propanil
BOH-Propanil
Cl
HO-N-H
Cl
3,CDichloroaniline
WA)
Cl
Cl
Cl
N-OH-DCA
6-OH-DCA
FIG.2. Hepatic microsomal metabolism of propanil and 3,4dichloroaniline.
epoxide hydrolase were generously provided by Dr. Andrew Parkinson, Kansas University Medical Center,
Kansas City. [ l-‘4C]Naphthol (0.02 pCi/pmol) was purchased from Amersham Corporation
(Arlington
Heights, IL).
Pretrkatments
and Microsomal
Preparation
Adult male Sprague-Dawley rats (250-300 g) were injected ip once daily for 3 consecutive days with corn oil
(5 ml/kg), phenobarbital (80 mg/kg), propanil(lO0 mg/
kg), or 3,4dichloroaniline’(lOO
mg/kg). Other rats received a single ip injection ofTCDD (6.5 &kg), TCAOB
( 100 mg/kg), or TCAB ( 100 mg/kg). All compounds were
dissolved in corn oil except phenobarbital, which was dis
solved in isotonic saline. Rats that received TCDD,
TCAOB, and TCAB were euthanized 4 days after treatment, while rats that received corn oil, phenobarbital,
propanil, and 3,4dichloroaniline
were euthanized 24 hr
after the last injection. Livers were removed, and microsomes were prepared by differential centrifugation as described previously (Leakey et al., 1987). Protein concentration was measured according to the method of Lowry
et al. (195 1) with bovine serum albumin as the standard.
Microsomal
Assays
Microsomal metabolism of propanil and 3,4-dichloroaniline was quantitated as described previously (McMilIan et al., 1990). Microsomal cytochrome P450 and cytochrome bJ were determined by the method of Omura and
Sato ( 1964). NADPH-cytochrome P450 reductase activity was assayed using cytochrome c by the method of
Phillips and Langdon (1962). Monooxygenase activities
toward 7-ethoxyresorufm, 7pentoxyresorufin, and 7benzoxyresorufin were measured as described previously
(Leakey et al., 1989a). UDP-glucuronyltransferase activity toward 1-naphthol was measured radiometrically in
unactivated (native) and detergent-activated microsomes as described previously (Leakey et al.. 1989b).
Data were analyzed for statistical significance by Student’s t test.
Microsomal proteins were also subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) using a Bio-Rad Mini-Protean II dual slab
cell according to the method of Laemmli (I 970), with
minor procedural modifications (Ryan et al., 1979; Ryan
et al., 1980; Waxman and Walsh, 1983). The separating
gel was 0.75 mm thick and 5.5 cm long, and contained
7.5% acrylamide. Proteins were stained with Coomassie
blue R-250.
ENZYME
INDUCTION
BY PROPANIL
0.40
DERIVATIVES
105
0.60
Propanil (mM)
FIG. 3. Substrate-velocity curves for formation of the propanil metabolite 3,4dichloroaniline in microsomes from rats pretreated with phenobarbital. TCDD, TCAOB, TCAB, propanil. and 3,4-dichloroaniline.
*p < 0.05.
RESULTS
Efects of Pretreatment on the Microsomal
Metabolism of Propanil
Liver microsomes were prepared from
adult male rats pretreated with propanil, 3,4dichloroaniline,
TCAB, TCAOB, TCDD, or
phenobarbital
as described under Materials
and Methods, and used to examine the effects
of enzyme induction on propanil and 3,4dichloroaniline
metabolism.
Induction
of
specific drug-metabolizing
enzymes was also
examined in microsomes from rats pretreated with propanil, 3,4-dichloroaniline,
TCAB, TCAOB, TCDD, or phenobarbital.
Figure 3 shows a plot of velocity versus
substrate concentration
for formation
of
the propanil metabolite 3,4dichloroaniline.
These data indicate that acylamidase activity
toward propanil was not induced by any of
the pretreatments. In fact, TCDD, TCAOB,
TCAB, and phenobarbital
pretreatments
produced significant decreases in acylamidase activity (p < 0.05).
In contrast, hydroxylation
of propanil at
the 2’-position was induced 2.4-, 2.7-, and
3.6-fold relative to the control by TCDD,
TCAOB, and TCAB pretreatments, respectively (Fig. 4). 2’-Hydroxypropanii
formation
was only slightly increased by pretreatment
with propanil (1 S-fold) or 3,4-dichloroaniline ( 1.9-fold), and was slightly decreased by
pretreatment with phenobarbital. These data
are consistent with 3-methylcholanthrene-induced 2’-hydroxylation
of 4-chloropropionanilide reported by Kiese and Lenk (1973).
Aromatic ring hydroxylation of propanil at
the &position, which was a relatively minor
pathway of propanil metabolism (McMillan
et al., 1990), was not significantly induced by
any of the pretreatments (Fig. 4).
Eflects of Pretreatment on the Microsomal
Metabolism of 3,4-Dichloroaniline
Microsomal
N-hydroxylation
of 3,4-dichloroaniline
was induced approximately
threefold by TCDD, TCAB, and TCAOB
pretreatments (Fig. 5); and this was the major
metabolic pathway in these microsomal incubations. Pretreatment of rats with 3,4dichloroaniline
also increased N-hydroxylation,
106
MCMILLAN
F.$2
2
Q
0.60
p
.-2
E
1
0.40
g
0.20
0.00
Control
Pb
TCDD
ET AL.
TCAOB
TCAB
Propanil
3,4-DCA
Treatment
FIG. 4. Rates of formation(v) of the propanil metabolites 2’-hydroxypropanil(2’-OH)
and Ghydroxypropanil (6-OH) in microsomes from rats pretreated with propanil derivatives. Concentration of propanil was
1 mM. Rates of formation of the metabolite are expressed as means k SD (n = 3). *Significantly different
from control, p d 0.05.
twofold; however, neither propanil nor phenobarbital pretreatment induced the formation of this metabolite.
Hydroxylation
of 3,4-dichloroaniline
in
the 6-position was induced twofold by 3,4dichloroaniline
pretreatment (Fig. 5). This
0.8
0.6
0.0
Control
Pb
TCDD
TCAOB
TCAB
Propanil
3,4-DCA
Treatment
FIG. 5. Rates of formation (v) of the 3,4-dichloroaniline metabolites N-hydroxy-3,4dichloroaniline
(NOH-DCA) and Ghydroxy-3,4dichloroaniline
(6-OH-DCA) in microsomes from rats pretreated with propanil derivatives. Concentration of 3,4dichloroaniline
was 1 mM. Variation of duplicate incubations did
not exceed 1%.
ENZYME
INDUCTION
BY PROPANIL
DERIVATIVES
107
P45Ob and P450e (Lubet et al., 1985), was
slightly induced (2- to 4-fold) by propanil,
3,4dichloroaniline,
TCDD, TCAOB, and
TCAB pretreatments. In contrast, phenobarbital pretreatment increased PROD activity
approximately 8 1-fold.
Microsomes from pretreated rats were also
Efects of Pretreatment on the Induction of subjected to SDS-PAGE, and the results are
Microsomal Drug-Metabolizing Enzyme shown in Fig. 6. Pretreatment of rats with
Activities
TCAB or TCAOB produced an intensification of protein bands that comigrated with
cytochromes P45Oc and P45Od; their electroHepatic monooxygenase activities toward
substrates that are specific for different forms phoretic pattern was identical to that of
of cytochrome P450 were compared in miTCDD-pretreated
microsomes. In contrast,
crosomes from rats pretreated with TCAOB,
no induction of protein bands corresponding
TCAB, propanil, 3,4-dichloroaniline,
TCDD,
to cytochromes P45Oc and P450d was obor phenobarbital
(Table 1). TCAB and served in microsomes from the livers of rats
TCAOB pretreatments produced an increase pretreated with propanil or 3,4dichloroaniin the specific content of total microsomal cy- line.
tochrome P450; however, an increase in cytoFinally, the effect of the pretreatments on
chrome P450 content was not observed in hepatic microsomal
UDP-glucuronyltransmicrosomes from rats pretreated with propaferase (GT) activity toward 1-naphthol,
nil or 3,4-dichloroaniline.
Cytochrome bS which is induced by 3-methylcholanthrene
content was also slightly elevated by all of the (Falany and Tephly, 1983; Ulhich and Bock,
pretreatments. In contrast, NADPH-cyto1984), was determined.
Only TCDD and
chrome P450 reductase activity, which has TCAOB pretreatments significantly induced
been shown to be induced approximately
I-naphthol GT activity (Table 2). Although
twofold by phenobarbital pretreatment (Shep
TCAB pretreatment produced a small elevahard et al., 1983), was not increased by tion in 1-naphthol GT activity, it was not sigTCDD, TCAB, TCAOB, propanil, or 3,4- nificant. No increase in 1-naphthol GT activdichloroaniline
pretreatments.
ity was observed in microsomes from proTCDD, TCAOB, and TCAB pretreatpanil- or 3,4-dichloroaniline-pretreated
rats.
ments produced greater than loo-fold increases in 7-ethoxyresorufin-O-deethylase
DISCUSSION
(EROD) activity (Table I), which is catalyzed
primarily by cytochromes P45Oc and P450d
(Burke et al., 1985). Induction of EROD acPropanil is one of the most important hertivity was also observed with microsomes
bicides in rice-producing areas of the United
from propanil (3.5-fold)- and 3,4dichloStates (Bartha and Pramer, 1970; Brewster,
roaniline
(6-fold)-pretreated
rats. 7-Benzpersonal communication,
1985). Although
oxyresorufin-0-dealkylase
(BROD) activity,
propanil is acutely toxic at relatively high
which is catalyzed primarily by cytochromes
concentrations (Singleton and Murphy, 1973)
P450d and P450b (Burke et al., 1985), was this herbicide is apparently not genotoxic
induced approximately
IO-fold by TCDD
(Ambrose et al., 1972; McMillan et al., 1988).
and TCAOB pretreatments. TCAB, propanil,
However, propani1 has been reported to be
and 3,4dichloroaniline
pretreatments
in- contaminated
with TCAB and TCAOB,
duced BROD activity only 3-fold. 7-Pentwhich are structural and toxicological anaoxyresorufin-0-dealkylase
(PROD) activity,
logs of TCDD (Poland and Knutson, 1982).
which is catalyzed primarily by cytochromes
In addition, TCAB has been demonstrated to
pathway was increased less than twofold
in microsomes from rats pretreated with
TCDD, TCAB, or TCAOB, and no increase
was observed in microsomes from rats pretreated with propanil or phenobarbital.
108
MCMILLAN
ET AL.
TABLE 1
EFFECTSOFPRETREATMENTW~THPROPANILDERIVATIVESONINDUCT~ONOFHEPATIC
MICROSOMALCYTOCHROME P450 ENZYMEACTIVITIES
Treatment
Cytochrome
P450
(nmol/mg
protein)
Cytochrome
bs
(nmol/mg
protein)
NADPH-cytochrome
P450 reductase
(nmol/min/mg
protein)
Control
PB
1.02 f 0.04b 0.39 * 0.02
1.42’
0.43’
0.42 + 0.02
NDd
TCDD
1.48 t- 0.05’
0.58 ? 0.02r
0.40 f 0.05
TCAOB
1.55 f 0.01,
0.54 + 0.01’
0.32 -+ O.Oti
TCAB
1.40 +- 0.031
0.5 1 f 0.02/
0.38 f 0.03
Propanil
0.98 + 0.07
0.50 + 0.01’
0.44 f 0.02
3,4DCA
1.03+0.81
0.52 -c O.Oll
0.41 + 0.04
EROD”
(nmol/min/
mg protein)
PROD”
(pmol/min/
mg protein)
BROD”
(nmol/min/mg
protein)
0.05 + 0.00
0.13’
(2.5)e
9.5 I + 0.75J
(190)
4.98 + 0.3 If
(100)
5.90 f 0.471
(118)
0.18 f 0.02r
(3.5)
0.3 1 & 0.04-r
8. IO f 0.70
653’
0.03 -I-0.00
ND
(f-5)
(2)
0 EROD, 7-ethoxyresorutin-O-deethylase; PROD, 7-pentoxyresorufin-O-dealkylase;
dealkylase.
b Mean + SEM.
‘Mean of duplicate determinations from pooled microsomes.
d Not determined.
c Values in parentheses represent -fold increases over control.
fp < 0.05.
be an environmental
degradation product of
propanil (Bartha and Pramer, 1970; You and
Bartha, 1982). Since TCAB and TCAOB are
TCDD-type inducers of cytochrome P450, it
is conceivable that exposure to these compounds may alter the metabolism and toxicity of propanil.
Propanil is thought to produce methemoglobinemia
in mammals
during hepatic
clearance of the parent compound. In a previous study, we identified and quantitated the
major metabolites
of propanil and 3,4dichloroaniline
in rat liver microsomes (McMillan et al., 1990). The major metabolite of
propanil was 3,4dichloroaniline.
In the presence of NADPH, 2’-hydroxypropanil
and 6hydroxypropanil
were also detected. 3,4Dichloroaniline
was metabolized primarily
to 6-hydroxy-3,Cdichloroaniline
and N-hydroxy-3,Cdichloroaniline,
and these metabolites directly oxidized hemoglobin in isolated erythrocyte suspensions.
(81)
32.7 f 1.5’
(4)
30.7 f 2.5/
(4)
24.3 + 3.5/
(3)
18.0 Z!I4.2’
(2)
18.3 + 2.3/
0.28 AZ0.01’
(9)
0.30 ?I 0.04’
(10)
0.09 +- O.Olf
(3)
0.09 + 0.03J
(3)
0.08 t 0.02r
(3)
BROD, benzoxyresorufin-0
In this study we examined the effects of enzyme induction on the microsomal metabolism of propanil and 3,4-dichloroaniline
because it was conceivable that exposure to
TCAB and/or TCAOB may alter the metabolism and toxicity of propanil. Acylamidasecatalyzed hydrolysis of propanil, which has
been shown to be an initial requirement for
the manifestation
of methemoglobinemia
(Singleton and Murphy, 1973; Chow and
Murphy, 1975), was not induced by any of
the pretreatments. These results are consistent with the results of Singleton and Murphy
(1973), who showed that phenobarbital pretreatment
slightly enhanced propanil-induced methemoglobinemia
without affecting
acylamidase activity. Phenobarbital, TCDD,
TCAB, and TCAOB pretreatments decreased
propanil hydrolysis for reasons that are not
completely understood. However, this effect
may be related to the increase in specific cytochrome P450 content in these microsomes.
ENZYME
INDUCTION
BY PROPANIL
109
DERIVATIVES
P-45oc
P-450bld
P-4509
P-460aEH
FIG. 6. SDS-polyacrylamide gel electrophoresis of purified cytochrome P450 and epoxide hydrolase
(EH) standards (0.05 pg) and microsomal protein (5 rg) from rats pretreated with TCDD. TCAOB, TCAB.
propanil, and 3,4-dichloroaniline (DCA).
2’-Hydroxylation
of propanil was induced
approximately threefold in microsomes from
TCDD-, TCAB-, and TCAOB-pretreated
rats, and was slightly induced by 3,4dichloroaniline
pretreatment. These results
are consistent with the results of Kiese and
Lenk (1973), who showed that 2’-hydroxylation of 4-chloropropionanilide
was induced
by 3-methylcholanthrene
in rabbit liver microsomes. These investigators also showed
that 3’-hydroxylation of 4-chloropropionanilide was induced by phenobarbital.
In the
studies reported in this paper, 3’hydroxypropanil was not detected as a significant metabolite of propanil by rat liver microsomes.
These results suggest that there may be species differences in the capacity for aliphatic
hydroxylation of arylamides.
In a previous study we reported that oxidative metabolism of propanil accounted for
approximately
20% of the total metabolism
at low substrate concentrations (0.05 mrvt),
while greater than 95% of the total metabolism could be accounted for by amide hydro-
lysis at relatively high substrate concentrations (1.0 mM) (McMillan
et al., 1990). In
microsomes
from TCDD-,
TCAB-,
or
TCAOB-pretreated
rats, oxidative metabolism of propanil accounted for nearly 80% of
the total metabolism at a substrate concentration of 0.05 mM (data not shown). However,
at a propanil concentration of 1.O mM, amide
hydrolysis accounted for approximately 90%
of the total metabolism. Although 2’-hydroxylation is the major pathway of propanil metabolism at low substrate concentrations, this
metabolite is also a substrate for the acylamidase (data not shown), which may be an important route of metabolism for this metabolite in viva.
N-Hydroxylation
of 3,4-dichloroaniline
was induced approximately
threefold by
TCDD, TCAB, and TCAOB pretreatments,
and twofold by 3,4-dichloroaniline
pretreatment. 6-Hydroxy-3,4-dichloroaniline
formation was only slightly induced by TCDD,
TCAB, and TCAOB (less than twofold). Pretreatment with 3,4-dichloroaniline,
which
110
MCMILLAN
TABLE 2
EFFECTOF PRETREATMENT WITH PROPANIL DERIVATIVESONINDUCTIONOFI-NAPHTHOLUDP-GLIJCURONYLTRANSFERASEACTIVITY
Activity (nmol/min/mg
Treatment
Control
TCDD
TCAOB
TCAB
Propanil
3,4-DCA’
Native
0.56+0.17*
1.35 +0.91c(2.4)d
1.92+0.11’(3.4)
0.83 f 0.09
0.33 + 0.07
0.53 2 0.14
protein)
Activated”
5.88 + 1.02
15.42 + 1.03’(2.6)
21.60+ 1.65’(3.6)
9.31 * 0.93
5.57 + 0.38
6.77 + 0.90
“Native = unactivated. Activated = activated with
0.5% digitonin.
b Mean f SEM.
‘pO.05.
d Values in parentheses represent -fold increases over
control.
e 3,4-Dichloroaniline.
was also the substrate, produced the largest
increase in the formation of 6-hydroxy-3,4dichloroaniline.
From these results one might assume
that pretreatment with TCDD-like inducers
might enhance the toxicity of propanil; however, efforts to enhance the methemoglobinforming potency of arylamines in general
have largely been unsuccessful (Smith and
Olson, 1973). Singleton and Murphy (1973)
reported that phenobarbital
pretreatment
only slightly enhanced the toxicities of propanil and 3,4dichloroaniline.
This observation
may be explained by the lack of significant
induction of 3,4-dichloroaniline
N-hydroxylation by microsomes from phenobarbitalpretreated rats. Nevertheless, enzyme induction is a complex process, often involving the
alteration of detoxication pathways as well as
bioactivation
pathways. Thus, it cannot be
assumed that an inducer of microsomal Nhydroxylation will enhance the methemoglobin-forming ability of arylamines.
Comparison of hepatic microsomal drugmetabolizing enzyme activities revealed that
TCAB and TCAOB were TCDD-type inducers, as expected. These results were confirmed
ET AL.
by SDS-PAGE. However, there appeared to
be differences in the ratio of cytochrome
P45Oc and P450d induction between TCAB
and TCAOB. The results from EROD and
BROD induction indicated that TCAOB had
induced more P450d than had TCAB. In addition, TCAOB significantly induced l-naphthol GT activity, while TCAB did not. It is
possible that the mechanisms of enzyme induction by these derivatives are slightly
different; however, differences in pharmacokinetics may be responsible for the observed
differences since TCAB has been shown to be
cleared more rapidly than TCAOB (Burant
and Hsia, 1984).
Propanil and 3,4-dichloroaniline
weakly
induced their own metabolism.
Although
these compounds did not increase the specific
content of P450, there were small increases in
EROD, PROD, and BROD activities. Although the patterns of induction by these
compounds resembled those of TCDD more
than phenobarbital,
we could not confirm
these results with SDS-PAGE;
however, a
three- to sixfold induction of these isozymes
may not have been sufficient to be observed
electrophoretically.
ACKNOWLEDGMENT
The authors thank Cindy Hartwick for the Preparation
of this paper.
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