Protoporphyrinogen Oxidase of Mouse and Maize: Target Site Selectivity and Thiol Effects on Peroxidizing Herbicide Action

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 57, 36–43 (1997) ARTICLE NO. PB972260

Protoporphyrinogen Oxidase of Mouse and Maize: Target Site Selectivity and Thiol Effects on Peroxidizing Herbicide Action Norman B. Birchfield and John E. Casida1 Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112 Received November 5, 1996; accepted March 20, 1997 The action of light-dependent peroxidizing herbicides involves inhibition of protoporphyrinogen oxidase (protox) at a high-affinity specific binding site readily assayed with our new N-aryltetrahydrophthalimide radioligand ([3H]THP), the desmethyl analog of flumipropyn. Protox of mouse liver mitochondria and maize etioplasts is similar in sensitivity to most of the 14 herbicides and analogs examined as inhibitors of [3H]THP binding, indicating that target site specificity is not a major factor in selective toxicity between mammals and plants. In assays using mouse protox, the 14 compounds fall into two groups upon correlating their ability to inhibit [3H]THP binding (without added thiol) and enzymatic activity [with added glutathione (GSH) or dithiothreitol (DTT) as an antioxidant for the substrate]. The inhibitory potency of the THPs in protox activity assays is reduced ,7-fold by GSH and ,200-fold by DTT relative to their potency in [3H]THP binding assays without added thiol. This “thiol effect” is only 2- to 4-fold with diphenyl ethers and oxadiazon. The reduction of THP potency by these thiols may be due to derivatization based on the identification of a N-aryl-cis-hexahydrophthalimide from incubation of flumipropyn with DTT. q1997 Academic Press

INTRODUCTION

available for the THPs in enzyme determinations or for any protox inhibitor based on radioligand binding assays. The THP desmethylflumipropyn was recently reported as a radioligand, referred to as [3H]THP, with high affinity and specificity for the inhibitor/herbicide binding site in solubilized preparations of mouse liver mitochondria (7). The specific binding site for this radioligand is validated as relevant to protox activity in two ways. First, there is a good correlation for five DPEs and two other herbicides between their potencies as inhibitors of [3H]THP binding and of protox enzyme activity (7). Second, oxyfluorfen administered ip to mice blocks [3H]THP binding in proportion to the dose with 50% inhibition at 4 mg/kg (7). In contrast to the enzyme activity assays in which the substrate is stabilized with a reducing agent such as dithiothreitol (DTT) or glutathione (GSH), the binding assay can be performed in the absence of added thiols (7) which greatly affect protox inhibition (8,9). This investigation compares the target site selectivity of peroxidizing herbicides, with emphasis on the THPs, at the [3H]THP binding site of

Protoporphyrinogen IX oxidase (proto2; EC 1.3.3.4) catalyzes the last common step in the biosynthesis of heme and chlorophyll and is therefore important in both animals and plants (1). Light-dependent peroxidizing herbicides including diphenyl ethers (DPEs) and N-aryltetrahydrophthalimides (THPs) (Fig. 1, showing structures, names, and numbers) are potent inhibitors of this enzyme (2), yet plants are much more sensitive than animals to their toxic effects (3). Mammalian and plant protox are similar in sensitivity to inhibition by four peroxidizing herbicides as determined in enzyme activity assays (4–6), but comparable information is not 1 To whom correspondence should be addressed. Fax: 510642-6497. 2 Abbreviations used: DMSO, dimethyl sulfoxide; DPEs, diphenyl ethers; DTT, dithiothreitol; GSH, glutathione; I50, concentration for 50% inhibition; proto, protoporphyrin IX; protogen, protoporphyrinogen IX; protox, protoporphyrinogen IX oxidase; PSCP, phenyl saligenin cyclic phosphonate (an esterase inhibitor); THPs, N-aryltetrahydrophthalimides; [3H]THP, the radioligand N-[4-chloro-2-fluoro-5-(propargyloxy)phenyl]-3,4,5,6-[3H]tetrahydrophthalimide.

36 0048-3575/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

PROTOX BINDING SITE SELECTIVITY AND THIOL EFFECTS

37

FIG. 1. Structures and names for peroxidizing herbicides and radioligand. Common names (and code numbers) are 1, acifluorfen-methyl; 2, oxyfluorfen; 3, nitrofen; 4, acifluorfen; 6, MK-129; 7, flumipropyn (S-23121); 8, desmethylflumipropyn (S-23142); 9, flumiclorac; 10, flumiclorac-pentyl (S-23031); 11, flumioxazin (S-53482); 12, chlorophthalim; 13, phenopylate; and 14, oxadiazon. Compound 5 is a nonherbicidal analog.

mouse and maize protox and considers the effects of thiols on mouse protox inhibition. MATERIALS AND METHODS

Chemicals [3H]THP (92 Ci/mmol, .99% radiochemical purity) was from our recent synthesis (7). Several herbicides and their analogs (1–6 and 12– 14) and esterase inhibitors (paraoxon and phenyl saligenin cyclic phosphonate or PSCP) were available from earlier studies in this laboratory. THPs 7, 8, 10, and 11 were provided by Valent U.S.A. (Walnut Creek, CA) and 9 was prepared by acid-catalyzed hydrolysis of 10 (4-hr reflux in water–acetic acid–concentrated hydrochloric acid, 58:39:3) followed by purification involving preparative silica TLC (Rf 0.32 with hexane–

ethyl acetate–acetic acid, 67:28:5). Other chemicals were obtained as follows: protoporphyrin IX (proto), GSH, and DTT from Sigma (St. Louis, MO); cis- and trans-1,2-cyclohexanedicarboxylic anhydrides and dodecyl-b-Dmaltoside from Aldrich (Milwaukee, WI). cis-N-[4-Chloro-2-fluoro-5-[(1-methyl-2propynyl)oxy]phenyl]hexahydrophthalimide was prepared by coupling the corresponding aniline with cis-1,2-cyclohexanedicarboxylic anhydride by a general procedure for N-aryl imides (10). Isolation in ,90% yield as an oil involved reversed-phase preparative HPLC (Beckman Ultrasphere, 5 mm ODS, 10 mm 3 25 cm column; gradient of 50% acetonitrile in water to 100% acetonitrile; retention time, 14.8 min). Molecular mass 349 Dal (fast atom bombardment mass spectrometry); 1H NMR (CDCl3) d

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BIRCHFIELD AND CASIDA

(ppm): 1.53 (4H, m), 1.71 (3H, d, 6 Hz), 1.93 (4H, m), 2.55 (1H, d, J 5 2 Hz), 3.09 (2H, m), 4.82 (1H, qd, J 5 7 Hz, J 5 3 Hz), 7.01 (1H, d, J 5 6 Hz), 7.29 (1H, d, J 5 9 Hz). The 1H NMR spectrum of this cis-imide was consistent with that of cis- but not trans-1,2-cyclohexanedicarboxylic anhydride. The trans-imide was not formed by reaction of the aniline with trans-1,2cyclohexanedicarboxylic anhydride under the same conditions. Mitochondrial and Etioplast Preparations Mouse liver mitochondria isolated as described by Brenner and Bloomer (11) were solubilized by stirring with sodium cholate (1% w/v) for 1 h at 0 8C. The suspension was centrifuged at 100,000g for 1 hr, and the clear brown– yellow supernatant was removed, rapidly frozen in liquid nitrogen, and stored at 2808C; no loss in activity of mouse protox was observed as a result of a single freezing. Captan-treated corn seeds (B73; Pioneer Hi-Bred International, Inc., Johnston, IA) were sprouted on vermiculite and grown for 7–9 days in the dark; the sprouts were exposed to fluorescent light for 4 hr and then to darkness for 2 hr before isolating etioplasts by the method of Pardo et al. (12) except deleting cysteine. Maize protox was solubilized with 1% (w/v) dodecyl-b-D-maltoside for 1 hr at 08C followed by centrifugation at 100,000g (13). Binding Assays Binding assays were performed with solubilized preparations of mouse mitochondria and maize etioplasts. The mouse binding assays involved incubating mitochondrial protein (180 mg) and [3H]THP (0.5 nM) in 0.5 ml of 100 mM phosphate buffer (pH 7.2). The ligand and inhibitors were added in dimethyl sulfoxide (DMSO) (15 ml), an amount held constant in all experiments. The mixtures were incubated for 15 min at 258C, and then 0.5 ml 60% saturated ammonium sulfate was added followed by an additional 15-min incubation before filtration through glass fiber filters (Whatman GF/C) and liquid scintillation counting (7). In a variation of these experiments, [3H]THP (0.5 nM) was

incubated with DTT (1 mM) in phosphate buffer (pH 7.2) for 5 to 80 min before addition of solubilized mitochondrial preparation and rapidly performing the binding assay. Specific binding was always determined as the difference between total and nonspecific binding in the presence of 0 or 500 mM acifluorfen, respectively. Inhibitor assay curves were analyzed by nonlinear least squares regression (7) to determine concentrations resulting in 50% inhibition (I50s). Maize binding assays performed as described (13) involved incubation of [3H]THP (0.5 nM), inhibitor(s), and solubilized etioplast preparation in 1 ml phosphate buffer as above for 30 min at 258C, followed by cooling in an ice bath for 10 min and filtration with polyethyleniminetreated GF/C filters. In one series of studies PSCP or paraoxon (100 mM) was included in the buffer with [3H]THP before adding solubilized etioplast preparation. Specific binding and inhibition curves were determined as for mouse protox. Enzyme Assays All enzyme activity assays were performed with solubilized mouse liver mitochondrial preparations. Protox activity was defined as the difference of proto production in the absence and presence of 3 mM oxyfluorfen, which completely blocks enzymatic but not background protogen oxidation. Protoporphyrinogen IX (protogen) was prepared by reducing proto in 20% ethanolic 10 mM potassium hydroxide and 66 mM sodium ascorbate using coarsely ground 5% sodium–mercury amalgam (7, 11, 14). The fluorometric assay conditions were as previously described (7). The reaction mixture (3.0 ml) consisted of 100 mM phosphate buffer (pH 7.2) with 1 mM EDTA, 0.2% Tween 20, 5 mM DTT, substrate solution (75 ml), inhibitor in DMSO or DMSO alone (10 ml), and mitochondrial preparation (10–50 ml). In assays without DTT, GSH was used as the antioxidant at a final concentration of 10 mM in 100 mM Tris buffer (pH 8.2) to reduce nonprotox substrate oxidation. DTT is much more effective than GSH for blocking

PROTOX BINDING SITE SELECTIVITY AND THIOL EFFECTS

nonprotox protogen oxidation such that background oxidation levels with DTT were negligible but with GSH could approach 25%. Functional enzyme assays were performed at room temperature by monitoring the linear region (r2 . 0.96) of proto fluorescence development over a 10-min period. RESULTS

Affinity and Selectivity of Inhibitors at the [3H]THP Binding Site of Maize and Mouse Five DPEs, seven THPs, and two other protox inhibitors were examined for potency in inhibiting [3H]THP binding in maize etioplasts and mouse liver mitochondria, allowing a selectivity factor to be calculated as the ratio of I50 values for maize versus mouse (Table 1). Two DPEs (1 and 2) and six THPs (6–11) have I50s of d5

TABLE 1 Potencies and Selectivities of Peroxidizing Herbicides as Inhibitors of [3H]THP-Specific Binding in Maize Etioplasts and Mouse Liver Mitochondria I50 (nM)b

Compound type and no.a

Selectivity Maize Mouse factorc

Diphenyl ethers 1 1.1 6.9d 2 3.3 1.8d 3 190 72d 4 370 85d 5 .50000 3000d N-Aryltetrahydrophthalimides 6 0.99 7.7 7 1.0 2.9 8 1.1 1.7 9 4.7 22 10 5.0 24 11 7.2 3.2 12 100 75 Other compounds 13 18 44d 14 86 32d a b c d

39

nM in one or both of the maize and mouse assays, whereas phenopylate (13) and oxadiazon (14) are less potent. Most of the compounds are relatively nonselective at the target site, although moderate specificity is indicated for the DPEs (except 1) toward mouse protox and for the THPs (except 11 and 12) toward maize protox. The apparent affinities of 9 and 10 are nearly identical under standard assay conditions (Table 1), but on adding paraoxon or PSCP as an esterase inhibitor to the buffer with solubilized maize etioplasts there is a four- to five-fold potency increase for flumiclorac-pentyl (10) in the binding assay (Fig. 2). Thiol Effects on Peroxidizing Herbicide Inhibition of Mouse Protox Activity Inhibitors (except 3) are less potent in protox activity assays with DTT (pH 7.2) than with GSH (pH 8.2) as the reducing agent (Table 2). The potency ratio comparing DTT with GSH averaged 2.7 6 1.4 for the DPEs versus 29 6 26 for the THPs. Oxadiazon (14) falls into the DPE range, but phenopylate (13) fits the THP pattern. These findings are also evident in the correlation lines between I50 values for [3H]THP binding and protox activity (Fig. 3).

0.16 1.8 2.6 4.4 .17 0.13 0.34 0.65 0.21 0.21 2.3 1.4 0.41 2.7

Compound numbers refer to Fig. 1. SE values averaged 23% of the tabulated I50s. I50 maize/I50 mouse. Data from Ref. 7.

FIG. 2. Effect of two esterase inhibitors on flumicloracpentyl inhibition of [3H]THP specific binding in solubilized maize etioplasts. Error bars are maximum and minimum values from duplicate determinations.

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TABLE 2 Effects of Dithiothreitol and Glutathione on the Potencies of Peroxidizing Herbicides as Inhibitors of Protox Activity in Mouse Liver Mitochondriaa I50 (nM)c Compound type and no.b Diphenyl ethers 1 2 3 4 5 N-Aryltetrahydrophthalimides 6 7 8 9 10 11 12 Other compounds 13 14

DTT

GSH

Potency ratiod

7.3 1.9e 3.7 0.92e 110 180e 520 170e 29000 16000e

3.8 4.0 0.62 3.1 1.8

200 77 200 11000 9900 370 38000

18 4.1 8.2 270 3700 4.5 1500

11 19 24 41 2.7 82 25

2900 140

170e 57e

17 2.5

a

Protox activity in the absence of inhibitors was 8.3 and 20.8 nmol proto formed/hr/mg protein for DTT and GSH, respectively. b Compound numbers refer to Fig. 1. c SE values averaged 16% of the tabulated I50s. d I50 with DTT/I50 with GSH. e Data from Ref. (7).

Correlations between Inhibition of [3H]THP Binding and of Protox Activity in Mouse The I50s of the THPs (6–12) for [3H]THPspecific binding correlate well with their I50s for protox activity and this is also the case for the DPEs and other herbicides (1–5, 13, and 14) (Fig. 3). However, the THPs fall on one correlation line and the other compounds on a second line such that the THPs are 10- to 30-fold less active as inhibitors of protox activity than anticipated from the binding data if all compounds behave in the same way. The correlation coefficient of binding I50 with protox activity I50 is 0.82–0.97 with each class of compounds alone, i.e., the THPs compared with the others as determined either with DTT or GSH, but much lower (0.46–0.75) for the two groups considered together, with GSH higher than DTT.

Effect of DTT and GSH on [3H]THP Binding in Mouse Thiol compounds in buffer lower the specific binding of [3H]THP compared with buffer alone. Thus, with 40-min preincubation of [3H]THP in phosphate buffer at pH 7.2, DTT (1 mM) essentially nullifies binding, whereas 15-min incubations of GSH (10 mM), mercaptoethanol (5 mM), or cysteine (5 mM) lower it by 22, 40, and 65%, respectively. However, GSH (10 mM) in Tris buffer at pH 8.2 lowers specific binding by 40%. By incubating [3H]THP with 1 mM DTT for increasing periods of time in the absence of mouse preparation and then quickly performing the binding assay it is evident that specific binding is inversely related to preincubation time with DTT (Fig. 4). Reactions of Flumipropyn and Phenopylate with Thiols Both flumipropyn (7) and phenopylate are decomposed in the presence of thiols. Incubating flumipropyn with GSH and phenopylate with DTT in phosphate buffer (pH 7.2) for 15 min results in 65–75% and 85–95% loss of the parent compound, respectively, as determined by HPLC with UV detection. Reaction of flumipropyn with DTT as above formed a new product evident by HPLC that was not present with GSH or no thiol under the same conditions. To characterize this product, the reaction was run on a larger scale by treating a solution of flumipropyn (78 mmol) with DTT (6 molar equivalents) in 56% acetonitrile in water (1.5 ml) for 6 days at 258C and then separating by reversed-phase HPLC. The major product (.90%) was specifically reduced at the cyclohexene but not the propynyl substituent since it was found to be identical to authentic cis-N-4-chloro-2-fluoro-5[(1-methyl-2-propynyl)oxy]phenyl]hexahydrophthalimide (Fig. 5) by HPLC, UV absorbance, FAB MS, and 1H NMR. Saturation of the flumipropyn double bond is a detoxification process based on mouse protox binding assay I50s of 3 and 47 nM for the starting material and reduced product, respectively.

PROTOX BINDING SITE SELECTIVITY AND THIOL EFFECTS

41

FIG. 3. Correlation for peroxidizing herbicides between I50s for [3H]THP binding and for protox activity in solubilized mouse liver mitochondria. Binding assays with no added thiol in phosphate buffer (pH 7.2). Protox activity assays with DTT (5 mM) in phosphate buffer (pH 7.2) or GSH (10 mM) in Tris buffer (pH 8.2). Data are from Tables 1 and 2. Correlation coefficient (r2) Thiol in protox assay DTT GSH

THPs (n 5 7)

Others (n 5 7)

THPs 1 others (n 5 14)

0.86 0.82

0.84 0.97

0.46 0.75

DISCUSSION

The potency of peroxidizing herbicides as inhibitors of [3H]THP binding is proportional to that for inhibition of protox enzyme activity, suggesting that with the DPEs (7) and THPs

FIG. 4. Effect of preincubation time with dithiothreitol on [3H]THP-specific binding to solubilized mouse liver mitochondria. The incubation time of [3H]THP and mitochondrial preparation in phosphate buffer (pH 7.2) was reduced to 5 min before adding ammonium sulfate and 5 min before filtration.

(this study) the binding interaction measured is the cause of enzyme activity block. However, the DPEs (plus oxadiazon) fall on one correlation line and the THPs (along with phenopylate) on another; the THPs are much less potent enzyme inhibitors in the presence of DTT than predicted from the binding results. This can be explained if the THPs and phenopylate react with thiols under the conditions of enzyme assay to form products of reduced potency as protox inhibitors. Several lines of evidence support this proposal for the THPs: DTT reduces the apparent binding of [3H]THP; replacement of DTT by GSH brings the correlation lines for binding versus enzyme inhibition closer together, possibly because GSH has a lower reactivity than DTT toward THPs; the THP moiety of the insecticide tetramethrin adds a thiol across the carbon–carbon double bond (15); the hexahydrophthalimide derived from flumipropyn and DTT in this study shows lower potency in binding assays; and the related N-alkyl maleimides are standard thiol-derivatizing agents. Phenopylate, although structurally distinct from the

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FIG. 5. Reaction of dithiothreitol with an N-aryltetrahydrophthalimide. The Michael adduct was not isolated.

THPs, also reacts with DTT, possibly via nucleophilic attack at the carbonyl. Inhibitor derivatization of a thiol in the active site of protox may be interesting speculation as earlier with tetramethrin at its neuroreceptor (15) but unlikely in the present case since thiol reagents do not inhibit mouse protox activity (16), [3H]THP binding is readily reversible (7), and some of the most potent protox inhibitors do not react readily with thiols. Thiols are important in the in vitro action of peroxidizing herbicides in three ways. They (a) slow nonprotox oxidation of substrate by minimizing autoxidation (17), (b) block enzymatic oxidation of protogen by herbicide-resistant plant oxidases (18, 19), and (c) react in several ways with inhibitors to alter their potency (20 and this study). These thiol effects have important ramifications. Many inhibitors, including [3H]THP, react with thiols, leading to low estimates of potency in enzyme assays which require these reducing agents; accurate potencies can be determined in the thiol-free [3H]THP binding assay. Our results show that DPEs are slightly more potent as inhibitors of protox activity in assays with GSH relative to DTT; however, this discrepancy may be due to the pH difference between the two assays. Factors other than target-site specificity must be major contributors to selectivity since mammalian and plant protox are similar in sensitivity to in vitro inhibition by peroxidizing herbicides. Metabolic activation or detoxification may take place with either the herbicides or accumulated tetrapyrroles. Flumiclorac as a carboxylic acid and its pentyl ester are similar in potency in binding assays but differ in lipophilicity and membrane penetration; the potency of the ester

in the present study was enhanced four- to fivefold by organophosphorus esterase inhibitors, suggesting that cleavage enzymes may contribute to selectivity and possible interactions with other pesticides. Differences in protogen oxidizing and degrading factors in animals and plants, in addition to the availability of light, may also confer selectivity. Peroxidizing herbicide action in plants involves other protogen-oxidizing factors insensitive to protox inhibitors, leading to accumulation of phototoxic proto in the cytoplasm (19, 21, 22). These protogen-oxidizing factors may be absent in animals since protogen accumulates in rat hepatocytes (23), thereby reducing excess proto production and its toxicity to animal cells. Proto accumulation could also be avoided if protogen-degrading factors are present in animals cells; this is analogous to a proposal for tolerant plants (24). In summary, although the light-dependent peroxidizing herbicides are more toxic to plants than mammals, they are relatively nonselective at their respective protox binding sites. ACKNOWLEDGMENTS The project described was supported by Grant PO1 ES00049 from the National Institute of Environmental Health Sciences, NIH, and by the University of California Toxic Substances Research and Teaching Program. We thank Allan Rose of Valent Technical Center (Dublin, CA) for samples of THPs and Tom Cromartie of Zeneca Agricultural Products (Richmond, CA) and Loretta Cole, Qing-Xiao Li, Nick Norberg, Gary Quistad, Edgardo Wood, and other colleagues of this laboratory for helpful suggestions.

REFERENCES 1. H. A. Dailey (Ed.), “Biosynthesis of Heme and Chlorophylls,” McGraw-Hill, New York, 1990. 2. S. O. Duke and C. A. Rebeiz (Eds.), “Porphyric Pesticides: Chemistry, Toxicology, and Pharmaceutical

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3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Applications,’’ American Chemical Society Symposium Series 559, Am. Chem. Soc., Washington, DC, 1994. C. Tomlin (Ed.), “The Pesticide Manual,” 10th ed., The British Crop Protection Council, The Bath Press, Bath, England, 1994. M. Matringe, J.-M. Camadro, P. Labbe, and R. Scalla, Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides, Biochem. J. 260, 231–235 (1989). M. Matringe, J.-M. Camadro, P. Labbe, and R. Scalla, Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: Oxadiazon, LS 82-556 and M & B 39279, FEBS Lett. 215, 38–38 (1989). A. V. Corrigall, R. J. Hift, P. A. Adams, and R. E. Kirsch, Inhibition of mammalian protoporphyrinogen oxidase by acifluorfen, Biochem. Mol. Biol. Int. 34, 1283–1289 (1994). N. B. Birchfield and J. E. Casida, Protoporphyrinogen oxidase: High affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria, Chem. Res. Toxicol. 9, 1135– 1139 (1996). J. M. Jacobs, N. J. Jacobs, S. E. Borotz, and M. L. Guerinot, Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria, Arch. Biochem. Biophys. 280, 369–375 (1990). B. Nicolaus, J. N. Johansen, and P. Bo¨ger, Binding affinities of peroxidizing herbicides to protoporphyrinogen oxidase from corn, Pestic. Biochem. Physiol. 51, 20–29 (1995). H. Ohta, S. Suzuki, H. Watanabe, T. Jikihara, K. Matsuya, and K. Wakabayashi, Structure-activity relationship of cyclic imide herbicides. I. N-Substituted phenyl3,4,5,6-tetrahydrophthalimides and related compounds, Agric. Biol. Chem. 40, 745–751 (1976). D. A. Brenner and J. R. Bloomer, A fluorometric assay for measurement of protoporphyrinogen oxidase activity in mammalian tissue, Clin. Chim. Acta 100, 259– 266 (1980). A. D. Pardo, B. M. Chereskin, P. A. Castelfranco, V. R. Franceschi, and B. E. Wezelman, ATP requirement for Mg chelatase in developing chloroplasts, Plant Physiol. 65, 956–960 (1980). N. Mito, R. Sato, M. Miyakado, H. Oshio, and S.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

43

Tanaka, In vitro mode of action of N-phenylimide photobleaching herbicides, Pestic. Biochem. Physiol. 40, 128–135 (1991). J.-M. Camadro, M. Matringe, R. Scalla, and P. Labbe, Fluorometric assay of protoporphyrinogen oxidase in chloroplasts and in plant, yeast, and mammalian mitochondria, in “Target Assays for Modern Herbicides and Related Phytotoxic Compounds” (Bo¨ger, P. and Sandmann, G., Eds.), pp. 29–34, Lewis, Boca Raton, FL, 1993. I. H. Smith, E. J. Wood, and J. E. Casida, Glutathione conjugate of the pyrethroid tetramethrin, J. Agric. Food Chem. 30, 598–600 (1982). H. A. Dailey and S. W. Karr, Purification and characterization of murine protoporphyrinogen oxidase, Biochemistry 26, 2697–2701 (1987). N. J. Jacobs and J. M. Jacobs, Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis, Enzyme 28, 206–219 (1982). K. Retzlaff and P. Bo¨ger, An endoplasmic reticulum plant enzyme has protoporphyrinogen IX oxidase activity, Pestic. Biochem. Physiol. 54, 105–114 (1996). H. J. Lee, M. V. Duke, and S. O. Duke, Cellular localization of protoporphyrinogen-oxidizing activities of etiolated barley (Hordeum vulgare L.) leaves, Plant Physiol. 102, 881–889 (1993). T. Shimizu, N. Hashimoto, I. Nakayama, T. Nakao, H. Mizutani, T. Unai, M. Yamaguchi, and H. Abe, A novel isourazole herbicide, fluthiacet-methyl, is a potent inhibitor of protoporphyrinogen oxidase after isomerization by glutathione S-transferase, Plant Cell Physiol. 36, 625–632 (1995). J. M. Jacobs, N. J. Jacobs, T. D. Sherman, and S. O. Duke, Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley, Plant Physiol. 97, 197–203 (1991). S. Yamato, Y. Suzuki, M. Katagiri, and H. Ohkawa, Protoporphyrinogen-oxidizing enzymes of tobacco cells with respect to light-dependent herbicide mode of action, Pestic. Sci. 43, 357–358 (1995). P. R. Sinclair, N. Gorman, H. S. Walton, J. F. Sinclair, J. M. Jacobs, and N. J. Jacobs, Protoporphyrinogen accumulation in cultured hepatocytes treated with the diphenyl ether herbicide, acifluorfen, Cell. Mol. Biol. 40, 891–897 (1994). J. M. Jacobs, J. M. Wehner, and N. J. Jacobs, Porphyrin stability in plant supernatant fractions: Implications for the action of porphyrinogenic herbicides, Pestic. Biochem. Physiol. 50, 23–30 (1994).