Chem. Res. Toxicol. 1996, 9, 1135-1139
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Protoporphyrinogen Oxidase: High Affinity Tetrahydrophthalimide Radioligand for the Inhibitor/ Herbicide-Binding Site in Mouse Liver Mitochondria Norman B. Birchfield and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112 Received April 26, 1996X
Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydrophthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 µg of protein) and [3H]THP (0.5 nM) are incubated in 500 µL of phosphate buffer at pH 7.2 for 15 min at 25 °C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 ) 0.97, n ) 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an ∼50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.
Introduction (protox),1
Protoporphyrinogen oxidase (EC 1.3.3.4) the last common enzyme in the heme and chlorophyll biosynthetic pathways, catalyzes the dehydrogenation of protoporphyrinogen IX (protogen) to protoporphyrin IX (proto). Many herbicides acting via peroxidation of lipid membranes are potent inhibitors of protox from plants and animals (1, 2). The human hereditary disease variegate porphyria is associated with half-normal protox activity (3) and results in an array of dermatological and neurological problems (4) and an elevated incidence of liver cancer (5). Test animals survive high doses of selected protox inhibitors, but in subacute or chronic feeding studies, these compounds increase porphyrin levels in feces and blood and carboxylated porphyrins in liver of rats and mice, much the same as occurs in variegate porphyria in humans (6, 7). Rats given single or chronic doses of many protox inhibitors show devel* To whom correspondence should be addressed, at the Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 114 Wellman Hall, University of California, Berkeley, CA 94720-3112; phone: (510) 6425424; fax: (510) 642-6497; e-mail address:
[email protected]. X Abstract published in Advance ACS Abstracts, September 1, 1996. 1 Abbreviations: the aniline, 4-chloro-2-fluoro-5-(propargyloxy)aniline; DHPA, 3,6-dihydrophthalic anhydride; EtOAc, ethyl acetate; I50, concentration for 50% inhibition; HOAc, glacial acetic acid; LSC, liquid scintillation counting; MeOH, methanol; Pd/C, palladium on activated carbon; proto, protoporphyrin IX; protogen, protoporphyrinogen IX; protox, protoporphyrinogen oxidase; THP and [3H]THP, N-(4chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydrophthalimide (also referred to in the literature as S-23142) and its 3H-labeled form as a radioligand; THPA and [3H]THPA, 3,4,5,6-tetrahydrophthalic anhydride and its labeled form.
S0893-228x(96)00074-4 CCC: $12.00
opmental effects including embryotoxicity, growth retardation, and teratogenicity (skeletal and cardiovascular) (8-10), and in one study fetal proto was greatly elevated (11). Extensively-studied protox inhibitors include the nitrodiphenyl ethers (e.g., acifluorfen and oxyfluorfen) and the even more potent tetrahydrophthalimides (12). The optimization of these and other herbicides as protox inhibitors has enabled the development of radioligand probes which have been successfully applied to plant protox (13-16), but there is no comparable report for mammalian protox. A suitable radioligand probe for protox in mammals must have both high affinity and high specific activity. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydrophthalimide (THP) was selected for study for three reasons: it has outstanding herbicidal activity and inhibits plant protox by 50% at 7 nM (14); it is a single isomer of relatively simple structure; tritium might be introduced into the tetrahydrophthalimide moiety at high specific activity. This paper describes the radiosynthesis of [3H]THP, the development of a binding assay, and validation of [3H]THP as a probe for the inhibitor/herbicide-binding site in protox from mouse liver mitochondria.
Experimental Procedures Synthesis of [3H]THP (Scheme 1). (A) Radiosynthesis Route. All reactions were initially optimized with hydrogen and deuterium under analogous conditions to those ultimately used with tritium. The radiosynthesis involved selective cata-
© 1996 American Chemical Society
1136 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Birchfield and Casida
Scheme 1a
a (a) NH OH, MeOH; (b) Pd/C, H or 2H , or 3H , EtOAc; (c) 4 2 2 2 HOAc, 110 °C.
Table 1.
1H
NMR Shifts for Environmentally-Sensitive Protonsa
compd
H-3
H-4
DHPA THPA THP [3H]THP
2.23 1.58 1.89 1.88
5.05 0.85 1.06 1.04
a C D with 5-mm probe. Bruker AM-400 spectrometer except 6 6 for [3H]THP with IBM AF-300 spectrometer. All signals are multiplets.
lytic reduction of the 4-ene of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation of labeled 3,4,5,6-tetrahydrophthalic anhydride ([3H]THPA) with 4-chloro2-fluoro-5-(propargyloxy)aniline (the aniline). The radiosynthesis involved only two chemical steps and one chromatographic purification. (B) Intermediates. Sources for the chemicals were as follows: DHPA from Frinton Laboratories (Vineland, NJ); THPA from Aldrich Chemical Co. (Milwaukee, WI); THP (98% purity) from Sumitomo Chemical Co. (Osaka, Japan). The aniline, previously reported from reduction of the corresponding nitro compound (12, 17), was prepared here by dissolving THP (350 mg) in a minimal amount of methylene chloride (CH2Cl2) and then diluting with methanol (MeOH) (30 mL), followed by slow addition of ammonium hydroxide (15 mL) until the solution became cloudy, standing for 30 min at room temperature, and evaporation with gentle heat under vacuum. Isolation involved dissolving the residue in MeOH and introduction into a silica gel column from which the aniline was eluted with CH2Cl2. Solvent evaporation gave the aniline as an oil which solidified after several days. Silica gel TLC yielded a single red spot following ninhydrin treatment of Rf 0.53 in hexane/ethyl acetate (EtOAc) 7:3 and 0.63 in CH2Cl2. The 1H NMR spectrum of the aniline [i.e., (CDCl3) δ (ppm): 2.54 (1H, t, J ) 2 Hz), 4.69 (2H, d, J ) 2 Hz), 6.55 (1H, d, J ) 8 Hz), 7.03 (1H, d, J ) 10 Hz)] was similar to the reported spectra for the aniline and THP (17). GC/MS using chemical ionization yielded the expected molecular mass. (C) Synthesis of [3H]THPA. Reduction of DHPA with hydrogen or deuterium gave THPA or [2H]THPA in 100% yield in 15 min with product characterization by comparison to authentic THPA by 1H NMR (Table 1) and IR spectroscopy (data not given). In the radiosynthesis, the reduction time was extended to 45 min (for potential isotope effects), and [3H]THPA was used directly for the next reaction. More specifically, DHPA (19.9 mg, 0.133 mmol) was dissolved in EtOAc (1 mL) with 10% palladium on activated carbon (Pd/C) (21.5 mg) and stirred under tritium gas (610 mmHg) for 45 min at room temperature. After tritium and some solvent were removed under vacuum, the residual solvent was frozen with liquid nitrogen and the remaining gaseous tritium removed under high vacuum. The flask was repressurized with nitrogen; the solution was thawed, then refrozen and placed under high vacuum again. This process was repeated one more time. EtOAc was then added to the solution which was transferred by needle to a 5-mL conical flask followed by a rinse of EtOAc. In the transfer step
the solution and rinse were passed through glass wool and a poly(tetrafluoroethylene) filter to remove Pd/C. Glacial acetic acid (HOAc) (0.5 mL) was injected into the conical flask, resulting in a faint blue-green color (not observed in preliminary experiments using hydrogen or deuterium). The solution was then evaporated to ∼0.1 mL followed by another addition of HOAc (0.5 mL). This process was repeated one more time to effectively remove EtOAc so the condensation step could be performed in HOAc. (D) Synthesis and Purification of [3H]THP. Attempts to condense the aniline with [1H]THPA were unsuccessful in EtOAc and mixtures of EtOAc and HOAc but successful in HOAc only. Accordingly, the aniline (9.3 mg, 0.0467 mmol) in 0.2 mL of HOAc was injected into the [3H]THPA solution (0.6 mL of HOAc) followed by two 0.1-mL rinses of HOAc. The solution was refluxed for 2 h at 110 °C (bath temperature)] (17, 18) in a nitrogen atmosphere, and then the solvent was stripped and the solid material lyophilized overnight. After adding freshly-distilled THF, the [3H]THP was purified on normalphase HPLC with a Merck LiChrospher column (10 cm, Si-60, 5 µm) using hexane-THF (88:12) as the mobile phase at 1.5 mL/min. UV absorbance was monitored at 285 nm and radioactivity with a β-ram detector. The total yield was 6.9 mg (0.0207 mmol, 45% based on the aniline), and the specific activity was 92 Ci/mmol determined by HPLC UV response and liquid scintillation counting (LSC). Mitochondrial Preparations. Mouse liver mitochondria (19) were solubilized by stirring with sodium cholate (1% w/v) for 1 h at 0 °C and centrifuging for 1 h at 100000g. The clear yellow-brown supernatant was removed (avoiding fatty deposits), rapidly frozen in liquid nitrogen, and stored at -80 °C. No loss in enzyme or binding activity was observed on frozen storage for up to 30 days at -80 °C. There was a rapid increase in binding activity for the first 90 min after thawing without further increase at 120 min (the recommended time for use in assays). Binding Assays. Binding assays were performed in 100 mM sodium phosphate buffer (pH 7.2) at 25 °C by sequential additions of 5 µL aliquots of Me2SO containing [3H]THP (0.5 nM final concentration in standard assays), competing inhibitor, and acifluorfen (none for total binding or 500 µM final concentration to measure nonspecific binding at each ligand or inhibitor concentration). Solubilized mitochondrial preparation (150 µg of protein) was added last in a 10 µL aliquot; then the mixture was vortexed and incubated 15 min at 25 °C. Analysis involved addition of 60% saturated ammonium sulfate adjusted to pH 7.2 (500 µL), followed by vortexing the mixture and allowing it to sit for 5 min before filtering on a Brandel Model M-24R cell harvester (Gaithersburg, MD) through glass fiber filters (Whatman GF/C). The filters were then rinsed with three aliquots (2 mL) of cold 10 mM phosphate buffer (pH 7.2), placed in scintillation vials with 1 mL of Optiphase HiSafe 2 scintillation cocktail (Wallac OY, Turku, Finland), and soaked for at least 2 h before LSC. Specific binding was defined as the difference between total and nonspecific binding in the presence of acifluorfen. Inhibitors were assayed between 0.1 and 105 nM at 10-fold dilutions with duplicate samples in each series and two sets of experiments. The concentrations for 50% inhibition (I50 values) were based on the 1-mL volume after adding ammonium sulfate solution, recognizing thereby the continuing association and dissociation during this assay step. Inhibition curves were analyzed using an iterative nonlinear least-squares regression on Sigma Plot software (Jandel Scientific Software, San Rafael, CA). Enzyme Assays. The assay measures the formation of proto in incubations containing mitochondrial preparation and protogen, with and without inhibitor. Protogen was prepared by reducing proto by a described procedure (19, 20) with the following exceptions: proto (Sigma Chemical Co., Milwaukee, WI) was reduced with coarse 5% sodium mercury amalgam (ground in hot glassware under nitrogen for consistent quality even with high humidity); all solutions were previously degassed with nitrogen; the reduced solution was passed through a
Binding Site of Protoporphyrinogen Oxidase
Figure 1. 3H NMR spectrum of [3H]THP showing protondecoupled chemical shifts and integrals. IBM AF-300 spectrometer at 320 MHz using C6D6 and 5-mm probe. No other signals are observed. Millipore Millex-HV (0.45 µm) filter; the pH was adjusted to ∼10 with degassed 20% phosphoric acid. Whereas proto in solution is burgundy red, the protogen solution is clear and stable for several weeks when stored at -10 °C under paraffin oil. Fluorescence measurements showed little or no proto but upon oxidation (enzymatic or at low pH) the protogen solution produced the fluorescence and absorbance spectra of proto. The incubation pH of 8.2 was selected to minimize non-protox oxidation of protogen. For fluoresence assay (19, 21), the incubations were made in 3.0 mL of 100 mM Tris buffer (pH 8.2) with 1 mM EDTA, 0.2% Tween 20, and 10 mM GSH. To this solution were added Me2SO (10 µL) alone or containing inhibitor, mitochondrial preparation (675 µg of protein, 45 µL), and finally the above protogen solution (75 µL, 5 µM final assay concentration based on the amount of proto subjected to reduction). Proto was measured fluorometrically by exciting at 405 nm and monitoring at 635 nm for 15 min with a Perkin Elmer LS50B Luminescence spectrometer equipped with a rotating, four-cuvette-holding turret for multiple samples. Enzyme activity was defined as the difference between the rate of proto production in the presence and absence of 3 µM oxyfluorfen (which completely inhibits protox activity leaving only background oxidation which was 10-25% of the protoxcatalyzed rate). Inhibition curves for I50 values were analyzed as above. In Vivo Experiments. Oxyfluorfen was administered ip at doses of 2-64 mg/kg to male albino Swiss-Webster mice using Me2SO (25 µL/mouse) as the carrier vehicle. The mice were sacrificed after 19 h for preparation of liver mitochondria and assay of [3H]THP binding as above.
Results Synthesis of [3H]THP. [3H]THP was synthesized in 45% yield at 92 Ci/mmol. The product was characterized by comparison to authentic unlabeled THP (17) and had identical properties on HPLC (Rt 8.8 min), cochromatographed on two-dimensional TLC (Rf ) 0.40 in hexane/ EtOAc 7:3 and 0.43 in CH2Cl2), and also gave similar inhibition in the low nanomolar range in the protox activity assay. The radiochemical purity was >99% by two-dimensional TLC as above and autoradiography. The 1H NMR spectrum of [3H]THP was identical to that of the unlabeled standard except the tetrahydrophthalimide proton signals were reduced, verifying both the structure and 3H labeling positions. 1H NMR of [3H]THP (benzene) δ (ppm): 1.04 (4H, m), 1.88 (4H, m), 1.96 (1H, s), 4.04 (2H, s), 6.83 (1H, d, J ) 9 Hz), 6.93 (1H, d, J ) 6 Hz); splitting of the propargyl protons was not observed due to the low sample concentration. 3H NMR showed the insertion of approximately one tritium atom at carbons 3 and 6 for two at carbons 4 and 5 (Figure 1).
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1137
Figure 2. Binding parameters for [3H]THP in solubilized mouse liver mitochondria. Solubilized preparation (180 µg of protein) was incubated with 0.025-3.0 nM [3H]THP in phosphate buffer (0.5 mL) at pH 7.2 for 15 min at 25 °C followed by addition of 60% ammonium sulfate solution (0.5 mL) and filtration. Specific binding is defined as the difference between total binding and nonspecific binding (with 500 µM acifluorfen) at each ligand concentration. The inset plot shows linearity for the 0.025-0.18 nM range in which nonspecific binding is minimal. The Scatchard plot at the right is based on two experiments on different days and includes the data (circles) from the plot of specific binding on the left.
Development of [3H]THP Binding Assay. Intact mitochondria are not suitable giving apparent nonspecific binding of 90-100% (or even higher with a nonpolar competing inhibitor that may induce ligand precipitation) presumably due to partitioning of the apolar ligand into lipid membranes. This problem was solved by solubilizing the protox activity and [3H]THP binding site prior to the assays and by adding the water-soluble acifluorfen to determine nonspecific binding. Sodium cholate was used in solubilization for several reasons: it extracts nearly 100% of the protox activity from mitochondria; it does not appear to lower enzyme stability as described for other detergents (22); it does not wash the solubilized enzyme through the GF/C filter to the extent observed with other solubilizing agents. Poly(ethylenimine)treated filters are not suitable to harvest protox solubilized with cholate or several other ionic or nonionic detergents. Dilution of the cholate-treated preparation below the critical micelle concentration allows some specific binding to be collected on untreated filters, and this is improved by adding ammonium sulfate (23) to precipitate the solubilized enzyme reaching a maximum for specific binding with ammonium sulfate at around 30% saturation before harvesting. Specific binding is identical before and after ammonium sulfate addition, suggesting that it does not hinder binding or denature a significant amount of the enzyme. The binding assay, relative to the enzyme assay, is less sensitive to change in pH and does not require reducing or chelating agents. Binding Parameters for [3H]THP (Figure 2). The specific binding is saturable with Kd 0.41 nM and Bmax 0.40 pmol/mg of protein. Attempts to show inhibition of [3H]THP binding by protogen at levels in the range of its Km (5.6 µM) (24) were not successful, resulting instead in unacceptable levels of nonspecific binding. Under the standard assay conditions, specific binding is characterized as follows (data not shown): completely abolished by heating the preparation at 90 °C for 10 min; linear with protein level in the range of 10-200 µg; equilibrium achieved in ∼10 min at 25 °C (leading to 15 min as the standard incubation time); dissociation rapid and binding completely reversible using either unlabeled acifluorfen or oxyfluorfen for displacement. Correlation between I50 Values for [3H]THP Binding and Protox Activity (Figure 3). The inhibitory
1138 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Figure 3. Correlation for peroxidizing herbicides between potency for inhibiting [3H]THP binding and protox activity in solubilized mouse liver mitochondria. The inhibitors are commercial or candidate herbicides available from previous studies in this laboratory as follows: 1 oxyfluorfen, 2 acifluorfen methyl, 3 oxadiazon, 4 phenopylate, 5 nitrofen, 6 acifluorfen, and 7 (a nonherbicidal analog) 4′-fluoro-4-nitrodiphenyl ether. The mean I50 values plotted from both the enzyme and binding assays are from two experiments involving duplicate samples with average deviations of 21% and 32% of the mean, respectively. The protein level, buffer, and incubation time differ in the two assays as indicated in the text.
Figure 4. Effect of oxyfluorfen in vivo on [3H]THP binding activity in solubilized mouse liver mitochondria. Analyses made 19 h after ip treatments with oxyfluorfen are related to controls in the same experiment with carrier vehicle only. The mean value for the controls was 15 500 dpm specific binding/mg of protein. Each point is based on two to four mice involving triplicate samples. Error bars represent the standard error of the mean (n ) 6-12).
potency of the nitrodiphenyl ethers (1, 2, and 5-7) and oxadiazon (3) and phenopylate (4) for [3H]THP binding correlates well (r2 ) 0.97) with that for protox activity, suggesting that the [3H]THP binding site is the same as that causing inhibition of protox activity. Effect of Oxyfluorfen in Vivo on [3H]THP Binding Activity (Figure 4). Oxyfluorfen, 19 h after ip treatment, blocks [3H]THP binding in a dose-dependent manner with ∼50% inhibition at 4 mg/kg.
Discussion The radiosynthesis of [3H]THP from [3H]THPA should be applicable to radiolabeling many 3,4,5,6-tetrahydrophthalimides. Insertion of tritium at the 3 and 6 carbons of DHPA, possibly via a radical stabilized by the doublyallylic nature of these methylenes, increased the specific activity of the [3H]THP by ∼30 Ci/mmol beyond the
Birchfield and Casida
expected additions at carbons 4 and 5, making this procedure useful for synthesis of ligands with high specific activity. The favorable correlation with several types of inhibitors between the I50s for [3H]THP binding and protox activity has two implications. First, the [3H]THP binding site is intimately associated with enzymatic activity and may be the substrate or catalytic site based on competition studies for both plant and animal protox by enzymatic (rather than binding) procedures (1); unfortunately, high nonspecific binding of [3H]THP at micromolar or higher protogen concentrations precluded a direct test of this hypothesis. Second, a large variety of inhibitors bind at the same or a closely-coupled site in plants (15, 24), and this may also apply to mammals. The characteristics of mammalian protox reported here appear to differ somewhat from those of plant protox based on literature cited above. Solubilized mouse protox, in contrast to solubilized maize enzyme (14), is not trapped on poly(ethylenimine)-treated filters, suggesting that mouse protox is less acidic than the maize enzyme. The Bmax of 0.40 pmol/mg of protein for the [3H]THP site in mouse mitochondrial protox in this study is 10- to 73fold lower than the value of 4 pmol/mg of protein for analogs of THP in maize etioplasts (14, 15) and 16-29 pmol/mg of protein for [3H]acifluorfen in corn and pea preparations (13, 24). This difference may be due in part to comparing heme biosynthesis as a basal rate in liver to a rapidly growing stage in plants. However, the protox concentration in mouse liver mitochondria is considerably higher based on relative enzyme activity determined during purification to homogeneity (25, 26) than on the Bmax in binding assays reported here, indicating the possibility of incomplete recoveries by the present procedure involving addition of ammonium sulfate and filtration. [3H]THP binding is a sensitive and convenient method for measuring inhibition of mammalian protox in vivo as illustrated in a dose-response experiment with oxyfluorfen. The inhibition measured suggests that oxyfluorfen or an active metabolite is present at the protox binding site or in the liver 19 h after treatment. It is clear that the binding assay has the requisite sensitivity to facilitate studies on the toxicology of other protox inhibitors as well. This is the first report of a binding assay for protox in mammals. [3H]THP appears to be highly potent and specific in quantitating this enzyme and should be useful in research on the mechanisms of heme biosynthesis and the toxicology of peroxidizing herbicides in plants and mammals.
Acknowledgment. The project described was supported by Grant PO1 ES00049 from the National Institute of Environmental Health Sciences, NIH, and the University of California Toxic Substances Research and Teaching Program. Tom Cromartie (Zeneca Agricultural Products, Richmond, CA) provided useful advice for the initial studies. We acknowledge our current or former laboratory colleagues Loretta Cole, Phillip Jefferies, Bachir Latli, Qing-Xiao Li, Gary Quistad, Andrew Waterhouse, and Edgardo Wood for useful suggestions and Nick Norberg for help performing protox activity assays. Hiromi Morimoto and Philip Williams of the National Tritium Labeling Facility (Lawrence Berkeley Laboratory, University of California at Berkeley) performed the radiosynthesis and 3H NMR analysis, respectively; this
Binding Site of Protoporphyrinogen Oxidase
facility is supported by NIH Grant RR01237, National Center for Research Resources.
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