Induction of cytochrome P450 4A1 in mouse liver by the herbicide synergist tridiphane

PESTICIDE

BIOCHEMISTRY

induction

AND

PHYSIOLOGY

of Cytochrome

44,

9-14 (1992)

P450 4Al in Mouse Liver by the Herbicide Synergist Tridiphane

PATRICIA E. LEVI, RANDY L. ROSE,NANCY H. ADAMS, AND ERNEST HODGSON Department

of Toxicology,

Box

7633,

North

Carolina

State

University,

Raleigh,

North

Carolina

2769s

Received February 25, 1992; accepted June 9, 1992 The herbicide synergist tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2,-trichloroethyl) oxirane] was examined for its ability to induce cytochrome P450 in vivo. Male C57BL/6N mice were given tridiphane, 250 mg/kg ip, for 3 days. Liver weight and P450 content were increased after tridiphane treatment, and SDS-PAGE showed an increase in microsomal proteins in the 50-60 kDa range. No significant increases in enzymatic activities were observed with the substrates benzphetamine, p-nitroanisole, benzo[a]pyrene, or ethoxyresorufin. There was, however, a IO-fold elevation in microsomal hydroxylation of lauric acid, an activity specifically associated with induction of P450 4Al. Western blot analysis using an antibody specific for P450 4Al showed a dramatic increase in P450 4Al. Q 1992 Academic Press. Inc.

INTRODUCTION

tive inhibitor of P450 activities in vitro (5). Assays with microsomes from phenobarbital and 3-methylcholanthrene-induced livers, as well as with purified P45Os 2B1 and 1Al in a reconstituted system, showed substantial inhibition of P450 2Bl activity, while P450 1Al activities were unaffected by tridiphane. Analyses of LineweaverBurke plots were consistent with competitive inhibition, with Ki values as low as 0.72 to 1.04 PM for p-nitroanisole O-demethylation. The inhibition data, along with spectral studies which yielded strong type I difference spectra with tridiphane and P450 2B1, suggest that tridiphane binds avidly to this P450 at or near the substrate binding site. These results, demonstrating tridiphane interactions with P450 in vitro, led us to examine the potential effects of tridiphane on P450 in viva.

Herbicides are among the most widely used pesticides, and currently available data suggest that as a class they are relatively nontoxic. Little is known, however, about their short- or long-term effects on mammalian systems. Tridiphane [2-(3,5dichlorophenyl)-2-(2,2,Ztrichloroethyl) oxirane] is registered as a postemergent herbicide and is used in formulations with atrazine as a herbicide synergist. Its mode of action as a synergist is at least partially due to the ability of tridiphane and its glutathione conjugate to inhibit the glutathione transferases responsible for the detoxication of atrazine in plants (1, 2). In addition, tridiphane has been shown to inhibit glutathione S-transferases in houseflies, thus synergizing the activity of certain insecticides (3). Although tridiphane has been shown to be a substrate for microsomal epoxide hydrolase in the mouse, and both a substrate and an inhibitor for glutathione S-transferases (4), very little is known about the interactions of tridiphane with mammalian cytochrome P450 enzymes (P450). Previous studies in this laboratory using microsomes and purified P450 isozymes demonstrated that tridiphane was a selec-

MATERIALSAND METHODS Chemicals

Chemicals used in the study were obtained from the following sources: benzphetamine, Applied Science (State College, PA); p-nitroanisole, Eastman Kodak Co. (Rochester, NY); ethoxyresorufin and resorufin, Pierce Chemical Co. (Rockford, 9 004%3575/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

10

LEVI

IL); benzo[a]pyrene, NADPH, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, anti-sheep IgG alkaline phosphatase, and NBT (nitro blue tetrazolium), Sigma Chemical Co. (St. Louis, MO); BCIP (5bromo-4-chloro-3-indolyl-phosphate), Boehringer-Mannheim Biochemicals (Indianapolis, IN); tridiphane (purity >99%), DOW Chemical Co. (Midland, MI); SDSPAGE reagents, Bio-Rad Laboratories (Piscataway, NJ); [I-i4C]lauric acid, Amersham (Arlington Heights, IL); and silica gel TLC plates from Brinkman Instruments (Westbury, NY). An antibody specific to cytochrome P450 4Al and purified cytochrome P450 4Al were generous gifts from Dr. G. Gordon Gibson (University of Surrey, UK). Treatment of Animals of Microsomes

and Preparation

C57BW6N male mice, 18-20 g (Charles River Laboratories, Raleigh, NC), were dosed intraperitoneally (six mice per group) for 3 days with tridiphane (250 mg/kg) in corn oil or corn oil alone. Animals were killed on Day 4 by CO, narcosis, and livers were removed and weighed. Livers from each treatment group were pooled and microsomes were prepared by differential centrifugation as previously described (5). Protein was measured by the fluorescamine protein assay (6) with bovine serum albumin as a standard, and cytochrome P450 content was determined from the dithionite-reduced CO difference spectrum (7). Enzyme Assays

Potassium phosphate buffer (100 mM), pH 7.6, containing 0.1 mM EDTA was used for all assays. Incubation mixtures contained 0.1-0.5 mg of microsomal protein, an NADPH-generating system (0.25 mM NADP+ , 2.5 mM glucose 6-phosphate, and 1 .O U glucose-6-phosphate dehydrogenase) and test substrate in a total volume of 1 ml. Benzphetamine N-demethylase activity was determined by measuring formaldehyde formation with the Nash reaction (8,

ET

AL.

9), ethoxyresorufin O-deethylase activity (10) and benzo[a]pyrene hydroxylase activity (11, 12) were determined spectrofluorometrically, and p-nitroanisole O-demethylation was quantitated by measuring formation of p-nitrophenol at 405 nm (13). Laurie acid hydroxylation was determined by separating [‘4C]lauric acid and metabolites on silica gel TLC plates using a hexane:ether:acetic acid (75:23.5: 1.25) solvent system as previously described (14) and quantitating on a Berthold linear analyzer. SDS-PAGE

and Western Blots

SDS-PAGE was performed according to the method of Laemmli (15) using a 7.5% polyacrylamide gel with a 3% stacking gel in a Bio-Rad Mini Protean II system (8 cm X 10 cm x 1 mm). Gels were electrophoresed under constant voltage (175 V) for 45 min. Gels were then either stained (0.1% Coomassie blue R-250 in 40% methanol and 10% acetic acid for 30 min) and destained (40% methanol and 10% acetic acid) or were electroblotted onto nitrocellulose paper by the method of Towbin et al. (16) at 100 V for 1 h in a Bio-Rad mini blot apparatus. Gels that were stained contained either 12, 16, or 21 u,g microsomal protein per lane. Fumarase (mol wt SO,OOO),catalase (mol wt 60,000), and bovine serum albumin (mol wt 68,000) were used as protein standards at a concentration of 1 )*g each. Gels for Western blotting contained 3 pg microsomal protein per land. Purified P450 4Al (0.1 pmol) was used as a standard. Nitrocellulose sheets with the transferred microsomal proteins were immunostained using a polyclonal antibody raised in sheep specific to rat P450 4Al as the primary antibody and anti-sheep IgG conjugated with alkaline phosphatase as the secondary antibody. The proteins were visualized by incubating the blots with 0.07 M BCIP (5bromo-4-chloro-3-indolyl phosphate) and 0.06 M NBT (nitro blue tetrazolium) in 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 0.04 M MgCl, at room temperature in the dark for 5-15 min.

INDUCTION RESULTS

Hepatomegaly was characteristic of the livers from the tridiphane-treated mice as indicated by an increase in liver weight and liver to body weight ratios (Table 1). A specific increase in cytochrome P450 content, measured as nanomoles of P450 per milligram of microsomal protein, also occurred. The values increased from 0.53 nmol/mg protein in the control microsomes to 0.92 nmol/mg protein in the microsomes from tridiphane-treated animals, suggesting induction of one or more P450 isozymes. SDS-PAGE analysis of microsomes from tridiphane-treated animals confirmed an increase over control in a number of microsoma1 proteins, especially in the 50-60 kDa region (Fig. 1). Enzyme activities characteristic of induction for specific P450 isozymes were selected for in vitro use to assess P450 induction. Results of these assays are shown in Table 2. Laurie acid hydroxylase activity, characteristic of P450 4A1, was increased IO-fold over control when expressed as activity/mg microsomal protein and 6-fold over control expressed as activity/nmol P450. When expressed as activity per nanomole of P450, the other measured activities were not increased, and in fact activity toward p-nitroanisole and benzphetamine declined (see Table 2). Western blot analysis of tridiphaneinduced microsomes using an antibody specific for P450 4Al showed a dramatic increase in a protein band corresponding to the same molecular weight as purified P450 4Al (Fig. 2), thus confirming induction of

1 C

of Tridiphane

on Liver

2 T

3 C

4 T

5 C

6 T

FIG. I. SDS-PAGE of hepatic microsomai proteins from control (C) and tridiphane-treated (T) mice. Lanes 1 and 2, 12 pg protein; lanes 3 and 4, 16 pg protein: and lanes 5 and 6, 21 kg protein.

TABLE Effect

11

OF P450 4A1 BY TRIDIPHANE

this P450 by tridiphane. The Western blot shows that this protein is also present, although at much lower levels, in the untreated animals. DISCUSSION

A number of chemically diverse compounds including certain hypolipidemic drugs, phthalate ester plasticizers, and phenoxyacetic herbicides (Fig. 3) have been shown to specifically induce P450 4Al as well as cause hepatic peroxisome proliferation in susceptible species (17-20). Tridiphane has been shown to cause peroxisome proliferation in mice and to induce microsomal epoxide hydrolase activity (21). The present study demonstrates that tridiphane also induces P450 4A1, suggesting that tridiphane acts in a manner analagous to the other peroxisome-proliferating agents. The chlorophenoxy herbicides, e.g., 2,4-D and 2,4,5-T, are structurally 1 Weight

and P450

Content

Treatment

Liver wt (g)

Liver wt/ body wt

P450 content (nmol/mg)

Control Tridiphane

1.37 1.87

0.19 0.25

0.53 0.92

Note. Male C57BL/6N mice were given tridiphane, 250 mg/kg ip, in corn oil for 3 days. Control animals received corn oil only. Livers from six mice were pooled and weighed prior to microsomal preparation. P450 values represent averages of triplicate determinations; values are within 10%.

12

LEVI

ET AL.

TABLE Effect

of Tridiphane

2

on Selected

P450 Enzyme

Activities

Activities per mg protein Substrate p-Nitroanisole Benzphetamine Benzo[a]pyrene Ethoxyresorufin Laurie acid

Control 2.22 8.18 1.03 0.09 1.85

k 2 2 2 2

per nmol P450 Tridiphane

0.37 0.36 0.15 0.12 0.57

2.21 12.3 2.03 0.27 18.4

2 ‘2 ? ‘-

0.67 0.45 0.25 0.05 4.08

Control

Tridiphane

4.18 15.4 1.94 0.18 3.49

2.41 13.3 2.21 0.29 20.0

Note. Values are expressed in nmol product/min; benzo[a]pyrene = fluorescent units/min. Methods are as for Table 1. Activities per nmol P450 were derived by dividing the mean activity per mg microsomal protein by the mean P450 value.

similar to the hypolipidemic drug, clofibrate, the prototype for peroxisome proliferation and P450 4Al induction, and therefore it is not surprising that they are inducers of P450 4A1. Tridiphane has a chlorinated phenyl ring but lacks the phenolic ether, the free carboxylic acid, and the ester moiety. Thus it represents another group of chemicals which causes peroxisome proliferation and induces P450 4Al. This finding suggests that other herbicides may also have this ability and should be examined. Cytochrome P450 4Al is known to be present in livers of untreated animals and is involved in the metabolism of lipids, specifically with the o-oxidation of medium- and long-chain fatty acids (22). This activity, coupled with the p-oxidation activities of the peroxisomal enzymes, is mechanistically related to lipid homeostasis and to the antihyperlipidemic effect of clofibrate and -

-

-

-

FIG. 2. Western blot of hepatic microsomal proteins from control (C) and tridiphane-treated (T) mice using an antibody specific to P450 4Al. Lane 1 V purified P450 4A1, 0.1 pmol; lanes 2 and 3, 3 kg microsomal protein.

similar drugs. Induction of P450 4Al by other inducing agents has been shown to involve increases in mRNA levels for P450 4Al beginning at 4-6 h after treatment with a subsequent synthesis of new enzyme (23). Although the mechanism by which these agents induce P450 and peroxisomes is not known, a receptor related to the steroid hormone family which binds a number of the hypolipidemic agents has been cloned recently (24). The exact relationship of this receptor to induction of P450 and peroxisome proliferation, however, is unclear. Although peroxisome proliferators are nonmutagenic in most in vitro tests, these chemicals induce liver tumors in rodents following chronic administration and may represent a separate class of epigenetic carcinogens (25). The mechanism(s) by which they exert their carcinogenic action is not known. Speculation has centered around the production of active oxygen species by the peroxisomes and subsequent DNA damage which may have an effect either in initiation, promotion, and/or progression of tumors (25). Although peroxisomal proliferation is an easily demonstrated, characteristic response of rodent livers to some hypolipidemic agents and related compounds, many other species show little or no response (20), and the susceptibility of humans in comparison to rodents is uncertain (25). For this reason it is particularly important to understand the mechanisms by

INDUCTION

OF

P450 4Al

BY

13

TRIDIPHANE

Tridiphane

CH3

Clofibrate

0-L-COOC,H,

Cl

LH,

Cl

2,4,-Dichlorophenoxyacetic acid (2,4-D)

0-CH,COOH

pH5

COOCH,CH(CH,),CH,

/-\ 8-O

Di-(2-ethylhexyl)phthalate DEHP

C2H3

CH3

0

-O-L-COOH

Nafenopin

AH,

FIG.

3. Structures

of selected

which these chemicals cause their toxicity, if these mechanisms (pathways) are operative in humans, and to what extent. Only then can the use of compounds, such as these pesticides and hypolipidemic drugs, be more effectively regulated and more safely used. Although tridiphane interacts with P450 both as an inhibitor and as an inducer, the actions are both isozyme specificinhibition of P450 2Bl and induction of 4Al. These findings serve to illustrate the importance of using purified isozymes and isozyme-specific antibodies, as well as microsomes, in the study of pesticide interactions with P450. ACKNOWLEDGMENT

This study is supported in part by PHS Grant ES(NIEHS).

00044

peroxisome

proliferators.

REFERENCES

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LEVI

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