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. REFERENCES AMBROSE, A. M., LARSON, P. S., BORZELLECA, J. 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