Aryl hydroxylation of diclofop by a cytochrome P450 dependent monooxygenase from wheat

PESTICIDE

BIOCHEMISTRY

AND

Aryl Hydroxylation

PHYSIOLOGY

34, 92-100 (1989)

of Diclofop by a Cytochrome Monooxygenase from Wheat’

JAMES J. MCFADDEN,

P450 Dependent

D. S. FREAR, AND E. R. MANSAGER

U.S. Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratoy, State university Station, Fargo, North Dakota 58105 Received December 9, 1988; accepted April 12, 1989 In tolerant wheat, diclofop metabolism in vivo was rapid (33% in 6 hr) and was sensitive to cytochrome P450 inhibitors (CO, tetcyclasis, and 1-aminobenzotriazole). Microsomal fractions from etiolated wheat seedling shoots were shown to support the in vitro hydroxylation of diclofop in the presence of molecular oxygen and NADPH. Enzyme activity was strongly inhibited by tetcyclasis, but showed less sensitivity to I-aminobenzotriazole and CO. The major enzymatic reaction product in vitro was isolated and identified by HPLC and electron impact mass spectrometry as the NIH shift product, 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxy]propanoic acid. The enzyme had a pH optimum of 7.4, a V,,, of 5.7 nmol/mg protein . h, and an apparent K, of 41.6 @cf.

8 1989 Academic

Press, Inc.

such as tetcyclasis or 1-aminobenzotriazole (6, 7). In the case of diclofop, the major hydroxylation product in vivo, 2-[4-(2,5dichloro-4-hydroxyphenoxy)phenoxy]propanoic acid, shows chlorine retention and migration (NIH shift) which is also consonant with the involvement of a cytochrome P450 (8). Two minor isomeric hydroxylation products of diclofop metabolism have also been identified in vivo (8). These minor metabolites include a second NIH shift product, 2-[4-(2,3-dichloro-4-hydroxyphenoxy)phenoxy]propanoic acid and 2-[4(2,4-dichloro-5hydroxyphenoxy)phenoxy]propanoic acid. There have been several reports of in fop (3). vitro hydroxylation of endogenous subAryl hydroxylation is an important method of herbicide detoxification and is strates (9-l 1). However, despite the imporcarried out by cytochrome P450 dependent tance of aryl hydroxylation in herbicide memonooxygenases (4, 5). In V&O character- tabolism, there have been few reports charization of metabolic dependence on cy- acterizing the enzymes involved. Makeev tochrome P450 enzymes is based on a re- et al. (12) reported the microsomal hydroxquirement for O2 and inhibition of metabo- ylation of 2,4-D[(2,4-dichlorophenoxy)lism by CO and cytochrome P450 inhibitors acetic acid] in peas and cucumbers. Cytochrome P450 enzymes are also known to support Wdemethylation of monuron in ’ Mention of trademark or proprietary products cotton (13) and aminopyrine in Jerusalem does not constitute a guarantee or warranty of the artichoke (14). product by the U.S. Department of Agriculture and General requirements for determining the does not imply its approval to the exclusion of other products that may also be suitable. involvement of cytochrome P450 enzymes INTRODUCTION

The herbicide diclofop-methyl [methyl(KS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propanoate] is one of a group of aryloxyphenoxy-propanoate herbicides used for postemergent control of wild oat and other grasses in cereal crops such as wheat (1). In both susceptible and resistant plants, diclofop-methyl is rapidly hydrolyzed to diclofop and herbicide selectivity is based on differential metabolism of diclofop (2). In wheat, diclofop is metabolized by aryl hydroxylation and O-glycosylation, while the primary metabolite produced in wild oats is a neutral glucose ester conjugate of diclo-

92 0048-3575189 $3.00 Copvright8 1989 by Academic All rights

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ARYL

HYDROXYLATION

as suggested by O’Keefe et al. (5) are (i) type of reaction products; (ii) requirement for reducing equivalents, O,, and inhibition by CO; (iii) spectral evidence for the existence of cytochrome P450 and substrate binding to cytochrome P450; and (iv) purification of catalytically active cytochrome P450. In this paper, we report the isolation of a microsomal fraction from etiolated wheat shoots that supports aryl hydroxylation of diclofop. The reaction characteristics meet the criteria suggested above, and the major reaction product cochromatographed with authentic 2-[4-(2,5-dichlorodhydroxyphenoxy)phenoxy]propanoic acid using HPLC. Electron impact mass spectrometry of the methylated reaction product also established that the dichlorophenoxy moiety was hydroxylated. MATERIALS

AND

METHODS

Plant material. Wheat seeds (Triticum aestivum L., cv. Olaf) were sown in ver-

miculite and irrigated with nutrient solution. After 6 days in darkness at 24°C etiolated wheat shoots (2.5-5 cm) were harvested and used for in vivo or in vitro assays. In vivo metabolism. Assays of diclofop metabolism in vivo were done using 1 g of etiolated wheat shoots in 50-ml Erlenmeyer flasks containing 2 ml of 25 mM MES (2-[N-morpholinolethanesulfonic acid) buffer, pH 6.2. Approximately 20 nmol of 2,4-dichlorophenoxy[U-i4C]diclofop (2 mCi/mmol) was added with various inhibitors and the flasks were incubated in a Dubnoff metabolic shaker at 30°C for 6 hr. After incubation, the segments were homogenized in 50 ml 80% MeOH with a Sorvall Omni-Mixer. Homogenates were vacuumfiltered to remove tissue debris and evaporated to dryness in vacua (40°C). Dried extracts were redissolved in 200 ~1 of 80% MeOH and aliquots were spotted on 250 p silica gel HF TLC plates (Analtech, Newark, DE). Chromatograms were developed in toluene:HOAc:EtOH (150:7:7), dried,

OF

DICLOFOP

93

and scanned using a Packard Model 7201 radiochromatogram scanner. Radioactive zones containing diclofop, OH-diclofop, and diclofop conjugates were scraped and counted in a Packard Tri-Carb scintillation spectrometer. Aglycones from in vivo assays were used as metabolite standards after glucoside hydrolysis with 1 N HCl at 50°C for 4 hr. Enzyme isolation and assay. All steps after harvest were done in a cold room at 4°C. Approximately 50 g of 6-day-old etiolated wheat shoots was harvested and floated in 10 mM Na2S,0S. Wheat shoots were then homogenized in a mortar and pestle with 1.5 vol of buffer containing 0.1 M K2HP04/KH2P04 (pH 7.4), 10% glycerol, 0.1% globulin-free bovine albumin (Sigma Chemical Co.), 5 mM DTT, and 1 mM Li,C03. Lithium was included to inhibit the breakdown of phosphoinositides (15). The buffer was degassed with N2 for 45 min prior to homogenization. The homogenate was filtered through four layers of cheesecloth and centrifuged in a Sorvall RC-2 centrifuge for 5 min at 1OOOgand 20 min at 6000g. The supernatant was then transferred to a Beckman LS-50 ultracentrifuge and centrifuged for 90 min at 100,OOOg.Pellets were resuspended in 4-8 ml of buffer (5-7 mg protein/ml) containing 10% glycerol, 50 mM KH2P04/K2HP04 (pH 7.4), and 1 mM mercaptoethanol. Aliquots were used for metabolism and spectrophotometric assays. Spectrophotometric data were obtained using a Beckman DU-7 equipped with a kinetics accessory. Cytochrome P450 difference spectra were obtained by the method of Estabrook and Werringler (16), using an extinction coefficient of 91 cm-’ mM- *. Cuvettes containing up to 2 mg protein in 1 ml of buffer were reduced with dithionite and a background spectrum was acquired. The sample cuvette was then bubbled with CO for 4 min and a CO difference spectrum was acquired. NADPH cytochrome P450 reductase was measured by the reduction

94

MCFADDEN,

FREAR,

of cytochrome c, according to the method of Strobe1 and Dignam (17) using an extinction coefficient of 6.2 cm-’ mM-‘. Substrate binding difference spectra were measured by a modification of the procedure of Jefcoate (18). Various substrates dissolved in DMSO to a concentration of 0.01 M were added to 1 mg of microsomal suspension after a background scan was obtained. DMSO alone was added to control cuvettes. Less than 10 ~1 total substrate solution (1%) was added to cuvettes in sequential measurements. Enzyme assayswere conducted using 0.5 ml of microsomal suspension (2-3.5 mg protein) and 1 mM NADPH in a total volume of 1 ml. Substrate 2,4-dichlorophenoxy[U-‘4C]diclofop (2 mCi/mmol) or [4-(2,4-dichlorophenoxy)phenoxy[U-3H]diclofop (4.9 CYmmol) and various cofactors or inhibitors were incubated with the enzyme for 10 min prior to starting the reaction by the addition of NADPH. Oxygen dependence was studied by conducting assays in septum-capped vials. Using a syringe, the vials were evacuated and filled with nitrogen five times prior to conducting the assays. The effect of carbon monoxide was assayed similarly, except that the vial was refilled with a mixture of 50% CO and 50% air. Assay mixtures were incubated at 30°C in a Dubnoff metabolic shaker for 1hr. Enzyme assays with [i4C]diclofop were used for metabolite identification, while high specific activity [3H]diclofop was used for enzyme kinetic studies. Enzyme reactions with [i4C]diclofop were stopped by the addition of 1N HCl to reduce the pH to 3.0. Acidified reaction mixtures were then extracted three times with 3 ml of ethyl acetate and the ethyl acetate was evaporated to dryness at 40°C with NZ, Extracts were then redissolved in 80% MeOH and analyzed by TLC (as above) or by HPLC. HPLC was performed with a Waters 5-mm Nova pack cartridge column and a 16-min linear gradient from 30% acetonitrile:water to 100% acetonitrile

AND

MANSAGER

at 1.4 mYmin. Both solvents contained 1% HOAc. The uv absorption was monitored at 254 nm on a Varian Vat-i-Chrom UV-VIS monitor and radioactivity was measured using a Packard Trace 7140 radioactivity monitor (RAM). Enzyme reactions containing r3H]diclofop were stopped by freezing in dry iceacetone. After the reaction mixtures were frozen, 0.25 g of Dowex-1X8 anion exchange beads (acetate form) was added to each reaction vial, and the reaction mixtures were allowed to thaw. The anion exchange beads adsorbed the unreacted [3H]diclofop and any hydroxylated [3H]diclofop that was formed. The reaction was followed by measurement of 3H20 formed by 3H displaced from the 2,4-dichlorophenoxy[U-3H]ring during oxygen insertion (17). The R- and S-enantiomers of [3H]diclofop were separated by isocratic HPLC on a Chiracel OD column (Daicel Chemical Industries, Ltd., Tokyo) using hexane:2propanol:acetic acid (80:20:1 v/v) at 1 ml/min. Baseline separated R (Rt = 5.2 min)- and S (Rt = 4.4 min)-enantiomers were collected and used in enzyme assays. Metabolite identification. Radioactive HPLC peaks from in vitro assayswhich had the same retention times as synthetic or in vivo 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxylpropanoic acid aglycone standards (Table 3) were collected and derivatized with methanolic HCl for 4 hr at 40°C (Instant Methanolic HCl, Applied Science, State College, PA). Under the HPLC conditions used, 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxy]propanoic acid readily separated (baseline) from isomeric synthetic standards of the two minor aglycones isolated from in vivo metabolism studies. After derivatization, samples were redissolved in 80% MeOH and rechromatographed by HPLC. The radioactive peak fractions were collected and analyzed by mass spectrometry. Electron impact spectra were obtained with a Varian MAT CH-

ARYL

5DF mass spectrometer ple probe. RESULTS

AND

HYDROXYLATION

using a solid sam-

DISCUSSION

In vivo metabolism studies. The effects of several cytochrome P450 inhibitors on diclofop metabolism in etiolated seedling shoot tissues are shown in Table 1. These data, together with the reported isolation and identification of NIH shift metabolites from diclofop-methyl-treated wheat shoots (8), strongly support the involvement of cytochrome P450 in diclofop metabolism. Cytochrome P450 isolation. The average cytochrome P450 content of etiolated wheat shoots was 0.21 nmol/mg protein. This is well within the range of reported values for plant tissues, but is roughly onetenth the amount present in rat liver tissue (Table 2). The generally low concentration of P450 in plant tissues may partly account for the diffkulty of purilkation and characterization of these enzymes (20). In some systems (11, 20), the levels of cytochrome P450 were increased by inducers such as phenobarbitol, ethanol, and 2,4-D. We were not able to show increased metabolism or increased levels of cytochrome P450 by pretreating tissues with 2,4-D or diclofop. Since we were not able to show induction and there was rapid metabolism in vivo (Table 3), it appears that metabolic activity TABLE 1 Effect of Cytochrome P450 Inhibitors on Diclofop Metabolism in Vivo % metabolism Control N2 co 10 p&f tetcyclasis 100 FM I-aminobenzotriazole

33.0 3.0 11.9 18.1

2 + * -t

% inhibition

3.0 0.2 4.4 0.6

91 64 45

24.1 ” 4.0

21

Nore. Etiolated wheat shoots (1 g fresh wt) were incubated with [‘4Cldiclofop. See Methods and Materials for details. Values are the average of two samples 57 variation.

OF

9.5

DICLOFOP

TABLE 2 Cytochrome P450 Content of Various Tissues Tissue

P450 content’

Wheat shoots Maize shoots Jerusalem artichoke Mung beans Tulip bulbs Rat liver

0.21 0.16 0.30 0.025 0.22 2.2

Ref. 30 30 19 20 31

a Units are nmoles cytochrome P45O/mg protein. All values are for crude microsomal preparations.

is associated with a constitutive cytochrome P450 enzyme system. Although cytochrome P420 levels in the crude microsome fractions were generally low (Fig. 1A), high levels of cytochrome P420 (up to 0.47 nmol/mg protein) were found in the supernatant fractions (Fig. 1B). Cytochrome P420 is considered to be a degradation product of cytochrome P450 (16, 21), and it is possible that low cytochrome P450 levels in plant microsomes are due to solubilization and conversion to TABLE 3 HPLC Retention Times of Reference 2-[4-(2,5-dichloro-4-hydro~phenoxy)pheno~]propanoic Acid and the Major Microsomal Reaction Product

.-

Retention time (min) Sample 2-[4-(2,5-Dichloro-4hydroxyphenoxy) phenoxylpropanoic acid” Methyl-2-[4-(2,5dichloro-4hydroxyphenoxy)phenoxy] propanoate” Aglyconeb Reaction product’ Methylated reaction product’

uv detector

-Radioactivity monitor

8.6

10.9 8.65 10.8-11.0

8.8-9.0 8.8-9.0 11.2

a Synthesized by Tanaka et al. (8). b Hydrolysis product of isolated major in vivo glycoside metabolite. ’ Microsomal oxidation product.

96

MCFADDEN,

-

380

FREAR,

AND

MANSAGER

A

410

440

470

500

530 Wavelength

380

410

440

470

500

530

(nm)

1. Reduced CO difference spectra. Observed diflerence spectra of wheat microsomes (A) and microsomal supernatant (B). Spectra were obtained by the method of Estabrook and Werringloer (16). FIG.

cytochrome P420 during homogenization. The addition of phenylmethylsulfonyl fluoride, NaF, EDTA, or KCN did not decrease cytochrome P420 formation below that obtained with bovine albumin. Substrate binding. Although a substrate binding difference spectrum is one of the criteria for cytochrome P450 involvement, the existence of additional pigments in plant microsomal preparations alters the absorption spectrum obtained. A typical binding spectrum for diclofop in wheat is compared with the expected spectrum in Fig. 2. Both curves show a minimum of

r-----l

0.05 I -0.05

380

410 Wavelength

440

470

500

lnm)

2. Diclofop-enzyme type I binding spectrum. DifJ*erence spectrum obtained by addition of diclofop in DMSO to microsomal suspension. Diclofop (-), expected curve (- - - - -). FIG.

42M25 nm, but the peak at 395 nm is rea duced. Repeated attempts to demonstrate a standard difference spectrum with endogenous substrates such as truns-cinnamic acid and lauric acid produced similar spectra (data not shown), suggesting that increased absorption at 395 nm is not a feature of substrate binding to crude microsomal cytochrome P450 preparations from plants. One possible explanation for this observation was reported by Higashi et al. (22) who found that oxidized tulip bulb cytochrome P450 was in the high spin form in the absenceof substrate. Since the increase in absorption at 395 nm is due to a shift in the spin equilibrium from low to high, if oxidized plant cytochrome P450 is primarily in the high spin state, little additional increase in absorption would be expected. Metabolite identification. Thin-layer chromatography of ethyl acetate extracts of enzyme assays consistently revealed low amounts of radioactivity in a zone that cochromatographed with hydroxylated diclofop aglycone standards. To further confirm the identity of the metabolite, extracts were chromatographed by HPLC. A typical HPLC chromatogram is shown in Fig. 3. The small peak with a retention time of 8.8 min cochromatographed with 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxy]pro-

ARYL

HYDROXYLATION

Retentlon

OF

Time

97

DICLOFOP

(mln)

3. HPLC of ethyl acetate soluble products from an in vitro assay. Microsomal reaction mixture was extracted with ethyl acetate, evaporated, and redissolved in 8Wo MeOHfor HPLC analysis. See Materials and Methods for chromatography conditions. FIG.

panoic acid, the aglycone of the major metabolite in vivo. The HPLC retention times of 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxylpropanoic acid standards and the major microsomal oxidation product are compared in Table 3. Retention times of the microsomal reaction products agree with the retention times of synthetic standards produced by Tanaka et al. (8). The major product from several microsomal reactions was collected, pooled, esterified with methanolic HCl, and analyzed 177

by mass spectrometry. The mass spectrum is shown in Fig. 4. Major ion fragments at m/z 297 (M-59) and m/z 269 (M-87) are typical for a methyl ester of propionic acid. In addition, the presence of the ion fragment at m/z 177 indicates that the 2,4-dichlorophenoxy moiety is hydroxylated. The radioactive peak on the HPLC chromatogram with a retention time of 10.5 min was also collected and was identified by mass spectrometry as the glycerol ester of diclofop. The formation of this product was

269

269

356(M)+

ci g

60

MW=356

0” f 0

f30-

3 2

150

FIG.

4. Mass

spectrum

2

3

L

250

300

350

J-----l 400

of merhylated microsomal oxidation product.

450

98

MCFADDEN,

FREAR, AND MANSAGER

not investigated in detail because it is not an oxidation product and is not formed in vivo. Small amounts of the glycerol conjugate were formed in NADPH or boiled controls, possibly by a nonenzymatic process similar to that reported by Smith (23). Enzyme assay and kinetics. Microsomal preparations from etiolated wheat coleoptiles supported low levels of diclofop hydroxylation in vitro and the enzyme characteristics were similar to that of a cytochrome P450 dependent monooxygenase. Resuspended pellet fractions were stable for up to 1 month when stored at -70°C. Hydroxylation was dependent on NADPH and 02, but was only slightly inhibited by carbon monoxide. No activity was observed in boiled controls or enzyme assays without NADPH. The low level of inhibition by CO may have resulted from the addition of substrate and NADPH before the addition of CO to the reaction. Cytochrome P450 inhibitors, tetcyclasis and laminobenzotriazole, also inhibited the enzyme (Table 4). These data, together with the identification of an enzymatic NIH shift reaction product, suggest that the hydroxylase is a cytochrome P450 monooxygenase. Based on double reciprocal plots obtained with substrate levels from 9.8 to 100 pA4, apparent Michaelis K,,, and V,,, values were estimated to be 44.1 @t4 and 5.7 p.mol/mg proteimhr, respectively. Enzyme activity was linear up to 3.8 mg protein. Comparison of activity in vivo and in vitro was done by calculating the average milligrams of microsomal protein per gram of TABLE 4 Effect of Cytochrome P450 Inhibitors on in Vitro Microsomal Enzyme Activity Inhibitor NZ co 10 FM tetcyclasis 100 fl 1-aminobenzotriazole

% inhibition” 78 12.5 84 42

+ 2 * +

14 5 8 10

LIValues are the average of at least three assays k standard deviation.

fresh weight tissue. The results suggest that 25-30% of the in vivo activity was recovered. NADPH-induced lipid peroxidation does not appear to contribute to the oxidation of diclofop since butylated hydroxytoluene, an anti-oxidant, and 3-methylindole, an inhibitor of lipid peroxidation (24), do not inhibit enzyme activity. Cytochrome P450 enzymes are known to discriminate between R- and S-enantiomers (25-27); however, we did not find significant differences in the hydroxylation of the diclofop enantiomers. Since it is known that the R-enantiomer of diclofop is herbitidally active (28), enantioselectivity must occur at the site of diclofop action, acetyl CoA carboxylase (29). Use of a high specific activity r3H]diclofop enabled us to accurately measure low levels of activity in substrate competition assays. As an indicator of their ability to tit the enzyme active site, several endogenous compounds and herbicides were tested as inhibitors of diclofop hydroxylation. None of the endogenous compounds tested (trans-cinnamic acid, kaurene, abscisic acid) were strong inhibitors of diclofop hydroxylation (data not shown). Weak inhibition by trans-cinnamic acid (1.8%) suggested that the cytochrome P450 responsible for diclofop hydroxylation may be different from the trans-cinnamic acid hydroxylase that was also present in the microsomal preparations. Several herbicides were strong inhibitors of diclofop hydroxylation (Table 5). AlTABLE 5 In Vitro Inhibition of Diclofop Hydroxylation Selected Herbicidesa Inhibitor 2,4-D Chlorsulfuron Haloxyfop Primisulfuron

by

% inhibitionb 30 100 98 92

a [3H]Diclofop concentration was 20 ~IW; herbicide inhibitor concentrations were 40 @4. b Values are the average of four assays.

ARYL

HYDROXYLATION

though 2,4-D is hydroxylated by a microsomal cytochrome P450 dependent reaction in higher plants (12), it was not a strong inhibitor of diclofop hydroxylation. It may be that the cytochrome P450 responsible for diclofop hydroxylation has a rather specific molecular size requirement and that 2,4-D is too small to effectively inhibit the enzyme. Chlorsulfuron, 1-(2chlorophenylsulfonyl)-3-(4-methoxy-6-methyl-1,3,5triazin2-yl)urea; haloxyfop, 2-[4-[3-chloro-5(trifluoromethyl-2-pyridinyl]oxy]phenoxy]propanoic acid; and primisulfuron, methyl-2-[3-(4,6-bis(difluoromethoxy)pyrimidin-2-yl)ureido-sulfonyl)benzoate, are all similar in molecular size to diclofop and all strongly inhibit diclofop hydroxylation. Whether these compounds are also hydroxylated by wheat microsomes was not tested. In summary, this report provides important preliminary data on a cytochrome P450 dependent monooxygenase responsible for diclofop hydroxylation in plants. Further study is necessary to purify the protein to homogeneity and to identify the physiological substrate(s) of the enzyme. A thorough characterization of the enzyme and the development of appropriate genetic probes should facilitate a better understanding of the role of monooxygenases in herbicide resistance and the development of strategies for combating resistant weeds. ACKNOWLEDGMENTS

The authors thank Mr. R. G. Zaylskie for his assistance in the mass spectrometry, Dr. R. H. Shimabukuro and Mr. H. R. Swanson for their helpful discussions, and Dr. F. S. Tanaka for the synthetic OHdiclofop standards. Diclofop was provided by Hoechst AG, primisulfuron by CIBA-Geigy Corporation, and chlorsulfuron by E. I. DuPont deNemours and Co., Inc. REFERENCES

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