J BIOCHEM TOXICOLOGY Volume 7,Number 2,1992

Effects of Diphenyl Ether Herbicides on Porphyrin Accumulation by Cultured Hepatocytes Judith M . JacoWA,Peter R. Sinclair#",Nadia Gorman",Nicholas J. Jacobs*, JacquelineF. Sinclair WilliamJ. Bement #, and Heidi Walton " #O,

Departments of *Microbiologyand "Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, 03756 and V A Medical Center, White River Junction, Vermont, 05009

ABSTRACT: Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted i n a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protoporphyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P45O-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.

Received November 1,1991. *To whom reprint requests should be sent. Address correspondence to Judith M. Jacobs, Department of Microbiology, Dartmouth Medical School, Hanover, NH, 037553842. Telephone: (603) 650-1665. 0 1992VCH Publishers, Inc.

Diphenyl Ether Herbicide, Acifluorfen, Acifluorfen Methyl, Protoporphyrinogen Oxidase, Porphyrin Accumulation, Cytochrome P450, Protoporphyrin, Coproporphyrin, Uroporphyrin.


INTRODUCTION Diphenyl ether (DPE) compounds of the general structure shown in Figure 1 are commonly used as commercial herbicides for the control of many types of weeds (1).The most important effect of DPE herbicides on plants is the light-induced breakdown of cell constituents by peroxidation (2). The mechanism by which DPEs cause light-induced lipid peroxidation resulting in cell disruption has recently been elucidated (3-5). DPE herbicides are powerful inhibitors of protoporphyrinogen oxidase (PPO), an enzyme in the heme and chlorophyll biosynthetic pathways which oxidizes protoporphyrinogen to protoporphyrin (3-5). Inhibition of PPO results in the accumulation of protoporphyrin, presumably by means of the slow, nonenzymatic oxidation of protoporphyrinogen to protoporphyrin in a manner that removes protoporphyrin from the biosynthetic pathways for heme and chlorophyll synthesis (6,7).Photo-excitation of protoporphyrin in the presence of oxygen results in the formation of reactive oxygen species, lipid peroxides and hydrogen peroxide, all of which are destructive to cell membranes (8). Little is known about the effects of DPE herbicides in mammalian systems. Although the acute toxicity of these herbicides for rats is low (9-ll), several reports indicate that these herbicides produce chronic toxicity (10-14). Although DPEs have not been examined for their ability to cause porphyrin accumulation in animals, recent studies have shown that several of these compounds inhibit mouse liver mitochondria1 PPO at concentrations similar to those that inhibit the plant enzyme (3,4). These results suggest that DPE herbicides may inhibit PPO in vivo and thereby cause 0007-2002/92/$3,50 + .25




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mulation in cultured hepatocytes; however, the pattern of accumulation is different in the rat and chicken cultures.



















FIGURE 1. Structure of Diphenyl Ether Herbicides.

porphyrin accumulation in animals as well as plants. To examine this question in animal systems, we investigated the effects of DPE herbicides on porphyrin ac&mulation in rat and chick embryo hepatocitecultures. These cultures have been frequently used as model systems to test the effects of drugs on regulation of heme and porphyrin synthesis in the liver (15, 16). The results of these experiments indicate that the DPE herbicides examined can cause porphyrin accu-

Acifluorfen (AF), technical (87%AF(sodium), 13% inert material), was a gift of Rhone-Poulenc Agricultural Chemicals, Research Triangle Park, NC. Acifluorfen methyl (AFM) (99.5%) was a gift of Stephen Duke, USDA, Stoneville, MS. Nitrofen (98%) was a gift from the EPA Pesticide and Industrial Chemical Repository, Research Triangle Park, NC.

Assay of Protoporphyrinogen Oxidation Barley etioplasts and liver mitochondria from 250 g Sprague-Dawley male rats and 16-day old White Leghorn chick embryos were prepared from tissue homogenates by differential centrifugation (17, 18).The isolated organelles were extracted with Triton X-100 (17). PPO activity was assayed in the Triton

TABLE 1. Effect of AFM and AF on Porphyrin Accumulation by Cultured Rat Hepatocytes. Hepatocytes in 6 cm dishes were prepared and cultured as described in the Methods. At 72 hr in culture, herbicides (dissolved in DMSO) were added with the medium change. At 93 hr in culture, cells and medium were combined and porphyrins determinedby HPLC as described in the Materials and Methods Section. Each culture dish contained 2 mg protein. Where indicated, 75 ph4 BNPP was added. The control culture was treated with DMSO alone. Addition of BNPP alone had no effect on porphyrin accumulationin otherwise untreated cultures (data not shown).The data represent means and ranges of duplicate plates. Treatment

Porphyrin (prnolehiish) Uro



Control + BNPP AF (14 pM) AF (14 pM) + BNPP

d.5 4 5 4.5

2*0 124 104

10a2 36*4 2822

AF (28 pM) AF (55 pM) AF (138 pM)

4.5 d.5 4.5

16tl 29*3 39*3

50*6 84+4 1204

AFM (14 pM) AFM (14 pM) + BNPP



144 47d

46*5 llla3

AFM (28 pM) AFM (28 pM)


d.5 5tl

164 70t7

45*4 120*4

AFM (55 pM) AFM (55 pM) + BNPP

4.5 104

32aO 79+14

92~2 172t6

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TABLE 2. Mefabolism of AF and AFM by Rat Hepatocytes. Hepatocytes in 6 cm dishes were cultured and treated as in Table 1. The final concentrations of AFM and AF after 21 hours in culture were determined on extracts of cells plus media by HPLC. Where indicated, 75 pM BNPP was added. Data are means and ranges of duplicate plates. ND: none detected Treatment

Herbicide added (pg /dish)

Herbicide Recovered (pg /dish)



Recovey AF 7 -+ AFM (%)

Control + BNPP AF (14pM) AF (14pM) + BNPP

0 17.5 17.5

0 16.0e0.8 17.2e1.1

0 0


0 91e5 9826

35.0 70.0 175.0

33.7k1.1 70.6tl.O 162.520.5

0 0 0

96*3 10121 9320

AFM (14pM) AFM (14pM) + BNPP

17.5 17.5

17.8t0.5 16.3e0.4

ND 1.920.5

102e3 10425

AFM (28pM) AFM (28pM) + BNPP

35.0 35.0

31.520.4 23.821.4

ND 11.622.5

90-1 101dO

AFM (55pM) AFM (55p M ) + BNPP

70.0 70.0

75.623.0 40.021.4

ND 28.620.7

10824 9823

AF (28pM) AF (55pM) AF (138pM)

extracts by a direct fluorometric assay developed by Camadro et ul. (19) as modified by Jacobs et ul. (20). Protoporphyrin (350 pM in 0.01 M KOH, 20% ethanol) was reduced to protoporphyrinogen with sodium amalgam, and was diluted 1:l with 0.5 M Tris HC1, pH 7.5, containing 50 mM DTT (21). A 10 pL aliquot was added to the assay mixture to yield final concentrations of 7 pM protoporphyrinogen and 1 mM DTT in the assay. The auto-oxidation rate of heatinactivated extract was determined for all conditions tested and was subtracted to determine the enzymatic rate shown (20). The auto-oxidation rates were less than 10%of the enzymatic rates and were not affected by AFM.

Hepatocyte Culture Hepatocytes from male Fischer F 344 rats (200 g) were prepared by perfusion of the liver through the venu ca'ou with EGTA followed by collagenase treatment according to the procedure of Bissell and Guzelian (22) as modified by Sinclair et al. (23). Viability of the cells was determined by Trypan Blue exclusion and was greater than 85%. Matrigel-coated culture dishes (6 cm) were inoculated with 3 million cells. Primary cultures from the livers of 16-day old White Leghorn chicken embryos were prepared as

described by Sinclair et al. (24). Specific treatments are described in the figure legends. The esterase inhibitor, bis-p-nitrophenylphosphate(BNPP) (Sigma), dissolved in saline, was added to some cultures to prevent the conversion of AFM to AF (15). Waterinsoluble chemicals were added in DMSO (maximum concentration 2 pL/mL media). For immunoblotting and determination of cytochrome P450, hepatocytes were solubilized with detergent as described previously (25).

Analysis of Porphyrins Cultured cells were scraped off plates into the culture medium and sonicated for 5 sec. An equal volume of acetone concentrated HC1(97.5/2.5, v/v) was added to a portion of the sonicate. The acid-acetone extracts were assayed for porphyrins by a modification of the reverse-phase HPLC method described by Bonkovsky et al. (26). Samples were diluted 1:l in Solvent A (56% 0.1 M NH4H2PO,, 44% CH,OH, pH 3.15). A 16-minute linear gradient was employed to increase Solvent B (100% methanol) from 40% to 100% at a flow rate of 1.1mL/min. A Waters p Bondapac CI8 column (3.9 X 300 mm) was used. Porphyrins were detected by their fluorescence with excitation at 398 nm and emission at 620 nm.



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Porphyrins were also determined by the direct spectrofluorometric method of Grandchamp et a1.(27) in which the fluorescence at three excitation/emission settings (400/595,405/595,410/605 nm) is measured and the three component porphyrins (uroporphyrin, coproporphyrin, protoporphyrin) determined with a matrix established from standard solutions of the porphyrins. For these analyses, porphyrins were extracted from the culture by adding an equal volume of 1 M HC107/methanol (1/1, v/v) to the culture medium. Results obtained with this method agreed with HPLC analysis of porphyrins in chick embryo cell cultures treated with DPEs. This method could not be used for rat hepatocyte cultures because unidentified porphyrins were falsely read as uroporphyrin.

Analysis for AF and AFM The amounts of AFM and AF remaining in the rat hepatocyte cultures were determined using the extraction procedure and HPLC method described above for porphyrin analysis, except that the absorbance at 313 nm was followed. Concentrations and retention times were determined from standard solutions of AFM and AF, In these cultures, HPLC analysis showed only two peaks following incubation: one with retention time of AFM and the other with retention time of AF.

Diphenylether (M)

FIGURE 2. Dose Responses of DPEs on rat liver PPO activity: Detergent extracts of rat liver mitochondria were prepared and assayed as described in the presence of 5 mM DTT as described in the Methods. Each data point represents the mean of two determinations. PPO activity without inhibitor was 10.3 1nmole/min/mg protein (0)AFM, (A) AF, (0)Nitrofen.

Analysis for Cytochrome P450 Spectral determinations of cytochrome P450 were performed by the method of Omura and Sat0 (28).The method of Towbin et aI. was used for immunochemical detection of cytochrome P450 isozymes (29) in detergent solubilized cells using 0.3% Tween 20 as the blocking solution (30). Preparation and characterization of the antibodies used are described by Sinclair et al. (31).

RESULTS Effect of AFM and AF on Porphyrin Accumulation by Rat Hepatocytes Table 1 illustrates the effects of acifluorfen in its free acid and methyl ester form in rat hepatocyte cultures. In the presence of AF, rat hepatocyte cultures accumulated coproporphyrin and protoporphyrin in a dose-dependent manner. The effect of AFM alone was equivalent to that of AF, but porphyrin accumulation was 2- to 3-fold higher at every herbicide concentration when conversion of AFM to AF was inhibited by the esterase inhibitor, BNPP (Table 1). This result indicates that AFM is more potent than AF in causing protoporphyrin accumulation. The cells

treated with both AFM and BNPP accumulated small amounts of uroporphyrin, but this was less than 10% of the total porphyrin present (Table 1).In two other experiments, the same general results were obtained, although the exact quantities of porphyrins accumulated at a given herbicide concentration varied from experiment to experiment. However, in all experiments the DPEs caused a dose-dependent increase in coproporphyrin and protoporphyrin without an increase in uroporphyrin. Table 2 shows the amounts of AF and AFM remaining in the cultures after 21 hr of incubation as determined by HPLC analysis (see Materials and Methods). More than 90% of the added AF was recovered at the end of the experiment (Table 2). In the absence of the esterase inhibitor BNPP, most of the added AFM was converted to AF during the incubation. In the presence of the esterase inhibitor, 11%to 41% of the methylated compound remained, with higher recovery at higher concentrations of AFM. These results indicated that rat hepatocytes readily converted AFM to AF and that BNPP partially prevented this hydrolysis. However, there was little further metabolism of AF.

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TABLE 3. Efect of AFM on Protoporphyrinogen Oxidase Activity of Detergent Extracts of Barley Etioplasts, Chick Emby o Liver Mitochondria, and Rat Liver Mitochondria. Triton X-100 extracts of sonicated organelles (etioplastsfrom 6-day old etiolated barley shoots, hepatic mitochondria from 16-day old White Leghorn chick embryos, and Sprague-Dawley rats) were assayed for PPO activity by the direct fluorometric assay. Enzyme preparations were preincubated for 20 min in the assay mixture with or without 5mM DTT (See Methods).The data represent means of duplicate assays, with individual values shown in parentheses. Protoporphyrinogen Oxidase Activity fnmole protoporphyrin formed/hr/mg protein) no DTT present 5mM DTT present no AFM AFM (20p M ) no AFM AFM (10 pM)

Barley Chicken Rat

43 (41,451 20 (1921) 22 (2123)

Effect of DPE Herbicides on Enzymatic Protoporphyrinogen Oxidation Table 3 shows that AFM inhibited enzymatic protoporphyrinogen oxidation in extracts of rat liver mitochondria, barley etioplasts, and chick embryo liver mitochondria. Preincubation of the enzyme with 5 mM DTT caused a decrease in PPO activity and markedly enhanced the AFM inhibition of rat and chick embryo PPO as previously reported for plant PPO (20). The effect of DTT and the inhibition by AFM were almost identical in extracts from rat and chick embryo liver mitochondria (Table 3). This effect of DTT in enhancing AFM inhibition of the liver enzyme has not been reported previously. Figure 2 shows the dose response of three DPE herbicides, (AF, AFM, or nitrofen) on PPO activity of rat liver mitochondria. Each of these herbicides inhibited at the 1 to 10 pM range with AFM being most potent. These findings are in agreement with previous reports showing that several DPE herbicides markedly inhibited mouse liver mitochondria1 PPO (3,4). Nitrofen had not been previously tested as an enzyme inhibitor.

Effect of AF or AFM on Porphyrin Accumulation and Cytochrome P450 Induction in Chick Embryo Hepatocyte Cultures Since protoporphyrinogen oxidation by chick embryo liver mitochondria was inhibited by AFM (Table 3), we determined whether AFM and AF could cause protoporphyrin accumulation in cultures of chick embryo hepatocytes. We compared these effects to those of the drugs, 2-propyl-2-isopropylacetamide



(29,31) 11

(19,21) 10


(9m 11 (10,12)

11 (10,12)

2 fL3) 2 (L3) 2 (L3)

(HA) and 3-methylcholanthrene (MC), which have been previously studied in the chick embryo culture system (15,16,32). In comparison with the other treatments, neither AF nor AFM alone increased the quantities of protoporphyrin or coproporphyrin present (Table 4).However, after treatment with AFM plus BNPP, massive amounts of uroporphyrin accumulated in the culture with perhaps a small increase in protoporphyrin. We also examined the ability of AF and AFM to induce cytochrome P450, because of the association between drug-induced uroporphyrin A 57 k D A









B 50 kDa









FIGURE 3. Immunochemical Detection of Isozymes of Cytochrome P450 Induced in Cultured Chick Hepatocytes by AF and AFM. Detergent solubilized cells from the chick embryo hepatocyte culture (Experiment 1) described in Table 4 were subjected to electrophoresis; the proteins transferred to nitrocellulose and immunochemically detected with either anti-P450,, (A) or anti-P-450G,,,(B)as described in the Materials and Methods section. Each lane contained 10 pg protein. Lane 1 contains protein from DMSO-treated hepatocytes; lane 2, PIA; lane 3, MC; lane 4, AF; lane 5, AF plus BNPP; lane 6, AFM; lane 7, AFM plus BNPP; lane 8, BNPP.



Volume 7, Number 2,1992

TABLE 4. Porphyrin Accumulation and Cytochrome P450 Induction in Cultured Chick Emby o Hepafocyfesafter Treatment with DPEs. Liver cells from 16-day old White Leghorn Chick Embryos were cultured as described in the Methods. After overnight treatment, cell plus media were assayed for porphyrins by direct spectrofluorometryas described in the Methods. Cytochrome P450 content was determined from CO-binding spectra (see Materials and Methods). The data represent means of 6 cm dishes from two separate experiments with individual values shown in parentheses. The values from Experiment 1are shown first. where indicated, BNPP was added at 30 pM, AF and AFM at 80 pM, PIA at 140 pM, and MC at 1pM. Treatment

Cytochrome P450 [pmole/mg protein)

Porphyrins [pmole/disk) Uro




45(20,70) 30(50,10)

40(80,0) 70(60,80)

55(8030) 65(60,70)

42(45,39) 51(33,69)


45(40,50) 40(50,30)

65(100,30) 60(80,40)

65(100,30) 70(100,40)

75(77,73) 76(85,67)

35(60,10) 1520(1920,1120)

80(90,70) 525(620,430)

85(100,70) 155(140,170)

78(97,59) 161(146,176)

50(60,440) 20(30,10)

45(50,40) 80(90,70)

175(50,300) 130(180,80)

148(140,155) 100(97,102)


accumulation and induction of specific cytochrome P450 isozymes in chick embryo hepatocyte cultures (16,32) Treatment with AFM plus BNPP caused a 4fold increase in cytochrome P450 (Table 4).The presence of the methyl ester on AFM appeared to be important for cytochrome P450 induction and uroporphyrin accumulation, since AF alone or AFM in the absence of the esterase inhibitor had minimal effects on cytochrome P450 or porphyrin accumulation. Massive accumulation of uroporphyrin is also observed when chick embryo hepatocyte cultures are treated with polyhalogenated aromatic hydrocarbons (PHAs)(16, 32). Since PHA-induced uroporphyrin accumulation by chick embryo hepatocytes has been associated with increases in MC-inducible isozyme(s) of cytochrome P450 (32), we identified some of the forms of cytochrome P450 present after treatment with AFM plus BNPP by immunoblotting with antibodies raised against two separate isozymes. Treatments with MC and PIA, chemicals known to induce different isozymes of cytochrome P450, were used as controls (31). Figure 3A shows that treatment with MC or AFM plus BNPP increased an isozyme of P450 (57 kDa) that cross-reacted strongly with the antibody raised against an MC-induced isozyme. This antibody did not crossreact with any PIA-induced isozymes (Figure 3A). Figure 3B shows that treatment with PIA or AFM plus BNPP increased a form of cytochrome P450 (50 kDa) which cross-reacted strongly with an antibody characterized as specific for an isozyme induced by the phenobarbital-like inducer, glutethimide (31). This antibody did not cross-react with the MC-induced isozyme

shown in Figure 3A, since no bands are visible at 57 kDa (Figure 3B). Thus, AFM plus BNPP had the unusual characteristic of inducing both an MC- inducible isozyme and a glutethimide-inducible isozyme, a property previously described for some nonplanar, polyhalogenated biphenyls (31).

DISCUSSION These experiments indicate that the DPE herbicides, AF and AFM, can cause porphyrin accumulation in animal hepatocytes, as has previously been reported for plants (3-5). In fact, the amount of protoporphyrin accumulated by rat hepatocytes after treatment with AFM is similar to that found in plants after treatment with the same chemical. Calculations based on the data reported here (Table 1) indicate that rat hepatocyte cultures accumulated about 40 nmoles total porphyrin (coproporphyrin and protoporphyrin) / g dry weight tissue after 21 hr treatment with 14 pM AFM plus BNPP. Matringe and Scalla (7) found that 60- 80 nmoles protoporphyrin accumulated / g dry weight of soy bean callous tissue after treatment for 14 hr with 10 pM AFM. The pattern of porphyrin accumulation observed after treatment of rat hepatocytes with AF or AFM was similar to that seen in the human disease, variegate porphyria (VP), which is characterized by elevated fecal coproporphyrin and protoporphyrin with no uroporphyrin accumulation (33). Patients with VP have a deficiency in the PPO enzyme, which is

Volume 7, Number 2,1992


assumed to be responsible for this pattern of porphyrin accumulation (34,35). Since we found that the PPO activity of rat liver mitochondria was inhibited at low concentrations of AF or AFM in an in vitro assay, we suggest that, by analogy to W, the accumulation of protoporphyrin and coproporphyrin found after treatment of rat hepatocyte cultures with AF or AFM was due to inhibition of PPO. (A different conclusion was drawn for the mechanism of uroporphyrin accumulation by avian hepatocytes as will be discussed below.) Although AF alone, as well as AFM alone, caused porphyrin accumulation in rat hepatocytes, the addition of BNPP to prevent conversion of AFM to AF clearly increased the effect of AFM. This result suggests that AFM is more potent than AF, a finding which agrees with the in vitro inhibition of PPO, where a lower concentration of AFM was required for maximum inhibition. This result further supports the inference that inhibition of PPO is part of the mechanism causing the porphyrin accumulation seen after treatment of rat hepatocytes with AF or AFM. DPE herbicides had a different effect on cultured chick embryo hepatocytes. The primary effect of AFM plus BNPP was to cause massive accumulation of uroporphyrin rather than protoporphyrin. These differences between chick embryo and rat hepatocyte cultures were not caused by differences in PPO, since in the in vitro assay, the PPO activities of rat or chick embryo hepatic mitochondria were equally sensitive to AFM. Apparently, any effect of AMF plus BNPP on protoporphyrin accumulation in chick embryo hepatocyte cultures was overwhelmed by the block earlier in the heme synthetic pathway. The different patterns of porphyrin accumulation observed in rat as compared to chick embryo hepatocyte cultures were most likely due to factors other than PPO inhibition, such as induction of cytochrome P450 isozymes and/or ALA synthase. The massive uroporphyrin accumulation caused by AFM plus BNPP treatment of chick embryo hepatocyte cultures was similar to that reported after exposure of these cultures to polyhalogenated aromatic hydrocarbons (PHAs) (32, 36). Recent studies have shown that PHA-induced uroporphyria in chick embryo hepatocyte cultures is associated with the presence of specific isozyme(s) of cytochrome P450 which mediate the oxidation of uroporphyrinogen to uroporphyrin (32,37). We found that the same treatment (AFM plus BNPP) which caused accumulation of uroporphyrin in chick embryo cultures, also increased the MC-inducible isozyme of cytochrome P450 (P450,) responsible for PHAinduced uroporphyrinogen oxidation (37). Treatment with AF or with AFM without the esterase inhibitor did not cause this massive uroporphyrin accumulation or increase cytochrome P45OMC,indicating the superior


inducing capacity of the lipid soluble ester in the chick system. These findings suggest that the mechanisms by which PHAs and DPE herbicides cause uroporphyrin accumulation in chick embryo hepatocytes are similar. We suggest that the rat hepatocyte cultures did not accumulate uroporphyrin, since they may not have been induced for cytochrome P450 IA2, the isozyme apparently required for uroporphyrinogen oxidation by rat liver microsomes (37). Rat hepatocyte cultures have been reported to be poorly inducible for this isozyme (38 and P. Sinclair,J. Sinclair, W. Bement and E. Schuetz, unpublished observations). Increased accumulation of porphyrins following treatment of chick embryo hepatocyte cultures with certain drugs has been associated with the induction of Saminolevulinic acid (ALA)-synthase, the first and the rate-limiting enzyme in the heme synthesis pathway (39). The accumulation of uroporphyrin caused by AFM plus BNPP (but not by AF or AFM alone) was probably also due, in part, to induction of ALA-synthase activity/although this was not examined directly. This treatment also induced the form of cytochrome P450 that has been associated with the induction of ALA-synthase (39,40). Thus, in cultured chick hepatocytes, treatment with AFM plus BNPP may have increased total porphyrin synthesis by induction of mediALA-synthase, followed by cytochrome P45OMC ated uroporphyrinogen oxidation, leading to the accumulation of massive amounts of uroporphyrin. In conclusion, we have shown for the first time that DPEs can affect the heme synthetic pathway in cultured hepatocytes. Different steps in the pathway are affected in rat and chick embryo hepatocytes, probably reflecting species differences in the inducibility of ALA-synthase and cytochrome P450 isozymes. Further studies will be needed to determine whether this group of herbicides will cause porphyrin accumulation in intact animals and thus have the potential to cause porphyria in humans.

ACKNOWLEDGMENTS This research was supported by funds from the U. S. Department of Veterans Affairs and National Institutes of Health (CA25012) and from the U. S. Department of Agriculture (Competitive Grant 9000705).

REFERENCES 1. K. I? Parry (1989). Herbicide use and invention. In Herbicides and Plant Metabolism, A. D. Dodge, ed., Cambridge University Press, New York, 1-20.


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2. K. J. Kunert, G. Sandmann, and P. Boger (1987). Modes of action of diphenyl ether herbicides. Rev. Weed Sci., 3, 35-55. 3. M. Matringe, J. M. Camadro, P. Labbe, and R. Scalla (1989). Protoporphyrinogen Oxidase inhibition by three peroxidizing herbicides: oxadiazon, LS 82-556 and M&B 39279. FEBS Lett, 245,3538. 4. M. Matringe, J. M. Camadro, P. Labbe, and R. Scalla (1989). Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides. Biochem. J., 260, 231-235. 5. D.A. Witkowski and B. P. Hallings (1988). Accumulation of photodynamic tetrapyrroles induced by acifluorfen methyl. Plant Physiol, 87,632-637. 6. J. Lydon and S. 0. Duke (1988). Porphyrin synthesis is required for photobleaching activity of the p-substituted diphenyl ether herbicides. Pestic. Biochem. Pkysiol., 31,7443. 7. M. Matringe and R. Scalla (1988). Studies on the mode of action of acifluorfen methyl in nonchlorophyllous soybean cells, Plant Physiol, 89,619-622. 8. F. R. Hopf and D. G. Whitten (1978). Chemical transformations involving photoexcited porphyrins and metalloporphyrins. In The Porphyrins, D. Dolphin, ed., Academic Press, New York, ~012,161-197. 9. W. 0.Johnson, G. E. Kollman, C. Swithenbank, and R. Y. Yih (1978). A new broad spectrum herbicide for post emergence use in soybeans. J. Agric. Food Chem., 26, 285-286. 10. A. M. Ambrose, P. S. Larson, J. F. Borzellica, R. B. Smith, and G. R. Hennigar (1971). Toxicologicalstudies on 2,4dichlorophenyl-p-nitrophenyl ether. Tox. Appl. Pharmacol., 19,263-275. 11. EPA (1987). Pesticide tolerance for lactofen. Fed. Regist., 52,10,567-10,568. 12. W. M. Draper and J. E. Casida (1983). Diphenyl ether herbicides: mutagenic metabolites and photoproducts of Nitrofen. I. Agric. Food Chem., 31,1227-1231. 13. W. M. Draper and J.E. Casida (1983). Diphenyl ether herbicides and related compounds: structure-activity relationships as bacterial mutagens. 1. Agric. Food Chem., 31,1201-1207. 14. E. G. Butler, T. Tanaka, T. Ichida, H. Maruyama, A.P. Leber, and G.M. Williams (1988). Induction of hepatic peroxisomes in mice by Lactofen, a diphenyl ether herbicide. Tox. Appl. Pharrnacol., 93,72-88. 15. G. Marks, B. Follows, D. Felt, and S. Cole (1983). Patterns of porphyrin accumulation in response to chemicals in chick embryo liver cells. Can. J. Physiol. Pharmacol., 61,546-553. 16. P. R. Sinclair, W. J. Bement, H. L. Bonkovsky, and J. F. Sinclair (1984). Inhibition of uroporphyrinogen decarboxylase by halogenated biphenyls in chick hepatocyte culture. Biochem. J., 222,737-748. 17. J. M. Jacobs and N. J. Jacobs (1987).Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and heme biosynthesis. Purification and partial characterization of the enzyme from barley organelles. Biochem. J., 244,219-224. 18. D. Johnson and H. Lardy (1967). Isolation of liver or kidney mitochondria. Methods Enzymol., 10,9696. 19. J. M. Camadro, N. G. Ibraham, and R. D. Levere (1985). Kinetic properties of the membrane-bound human liver mitochondria1 protoporphyrinogen oxidase. Arch. Biochem. Biophys., 242,206-212. 20. J. M. Jacobs, N. J. Jacobs, S. Borotz, and M. L. Guernot

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28. 29.







(1990).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. N. J. Jacobs and J. M. Jacobs (1982).Assay for protoporphyrinogen oxidation, a late step in heme synthesis. Enzyme, 28,206-217. D. M. Bissell and P. Guzelian (1980). Phenotypic stability of adult rat hepatocytes in primary hepatocyte cultures. Ann. N.Y. Acad. Sci., 349,85-87. P. Sinclair, W. Bement, S. Haugen, J. Sinclair, and P. Guzelian (1990). Induction of cytochrome P450 and 5aminolevulinate synthase in cultured rat hepatocytes. Cancer Research, 50,5219-5224. J. F. Sinclair, E. Smith, W. J. Bement, P. R. Sinclair, and H. L. Bonkovsky (1982). Increases in cytochrome P450 in cultured hepatocytes mediated by 3- and 4-carbon alcohols. Biochemical. Pharmacol., 31,2811-2815. J. F. Sinclair, P. R. Sinclair, and H. L. Bonkovsky (1979) Hormonal requirements for the induction of cytochrome P450 in hepatocytes cultured in a serumfree medium. Biochem. Biophys. Res. Commun., 86, 710-714. H. Bonkovsky, S. Wood, S. Howell, P. Sinclair, B. Lincoln, J. Healey, and J. Sinclair (1986).High performance liquid chromatographic separation and quantitation of tetrapyrroles from biologic materials. Anal. Biochem., 155,56-64. B. Grandchamp, J. Deybach, M. Grelier, and Y. Nordmann (1980). Studies of porphyrin synthesis in fibroblasts of patients with congenital erythropoietic porphyria and one patient with homozygous coproporphyria. Biochem. Biophys. Acta, 629,577-586. T. Omura and R. Sat0 (1964). The carbon monoxide binding pigment of liver microsomes. J.Biol. Chem., 230, 2370-2385. H. Towbin, T. Staehelin, and J. Gordon (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76,43504354. J. F. Sinclair, J. McCaffrey, P. R. Sinclair, J. W. Bement, L. K. Lambrecht, S. G. Wood, J. 8. Schenkman, and S.S. Park (1991). Ethanol increases cytochromes P450 IIE, 11B1/2, and IIIA in cultured rat hepatocytes. Arch. Biochem. Biophys., 284,360-365. P. R. Sinclair, J. Frezza, J. Sinclair, W. Bement, S. Haugen, J. Healey, and H. Bonkovsky (1989).Immunochemical detection of different isozymes of cytochrome P450 induced in chick hepatocyte cultures. Biochem. J., 258, 237-245. P. R. Sinclair, W. J. Bement, H. L. Bonkovsky, R. W. Lambrecht, J. E. Frezza, J. F. Sinclair, A. J. Urquhart, and G. H. Elder (1986). Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Biochem. J., 237,63-71. L. Eales, R. Day, and G. Blekkenhorst (1980). The clinical and biochemical features of variegate porphyriaAn analysis of 300 cases studied at Grute Schuur Hospital, Capetown, lnt. J. Biochem., 12,837-853. D. A. Brenner and J. R. Bloomer (1980). The enzymatic defect in variegate porphyria. N. Engl. J. Med., 302, 765-769. J. C. Deybach, H. de Verneuil, and Y. Nordmann (1981). The inherited enzymatic defect in porphyria variegata. Hum. Genet., 58,425428.

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36. P.R.Sinclair and S. Granick (1974).Uroporphyrin formation induced by chlorinated hydrocarbons (lidane, polychlorinated biphenyls, tetrachlorodibenzo-pdioxin). Requirements for endogenous iron, protein synthesis and drug-metabolizing activity. Biophys. Biochem. Res. Comm., 61,124-133. 37. J. M.Jacobs, P.R. Sinclair, W. J. Bement, R. W. Lambrecht, J. F. Sinclair, and J. A. Goldstein (1989).Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P450. Essential role of cytochrome P450-d.Biochem. J., 258,247-253. 38. A. R.Steward, S. A. Wrighton, D. A. Pasco, J. B. Fagan, D. Li, and I? S. Guzelian (1985).Synthesis and degradation


of 3-methylcholanthrene-induciblecytochromes P-450 and their mRNAs in primary monolayer c u l t u ~ sof adult rat hepatccytes.Arch. Biochem. Biophys., 241,494508. 39. S.Granick (1966).The induction in vivo of the synthesis of Gaminolevulinic acid synthetase in chemical porphyria: a response to certain drugs, sex hormones, and foreign chemicals. J. Bid. Chem., 241,1359-1375. 40. J. W. Hamilton, W. J. Bement, P. R. Sinclair, J. F. Sinclair, and K. E. Wetterhahn (1988).Expression of Gaminolevulinate synthase and cytochrome P450 mRNAs in chicken embrvo heuatocvtes in vivo and in culture. Effects of porphyroienic drugs and heme. Biochem. J., 255,267-275.

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes.

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll pre...
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