An investigation into the role of lipid peroxidation in the mode of action of aromatic hydrocarbon and dicarboximide fungicides

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

44,

91-100 (1992)

An Investigation into the Role of Lipid Peroxidation in the Mode of Action of Aromatic Hydrocarbon and Dicarboximide Fungicides ANN B. ORTH,* ANGELO SFARRA,~ EVA J. PELL,? AND MINGTIEN*,' *Department

of Molecular

and Cell Biology and University, University

tDepartment of Plant Pathology, Park, Pennsylvania 16802

Pennsylvania

State

Received March 20, 1992; accepted June 23, 1992 The mode of action of aromatic hydrocarbon and dicarboximide fungicides has been the subject of many studies which have not conclusively identified the primary target site. One current theory proposes that active oxygen species generated by these compounds initiate lipid peroxidation. We studied the effects of two aromatic hydrocarbons, chloroneb and tolclophos-methyl, and three dicarboximides, vinclozolin, iprodione, and myclozoline, on microsomes of Ustilago maydis. As a control, we compared the effect of paraquat, which is known to generate active oxygen, with that of these fungicides. Growth of U. maydis is very sensitive to all five compounds under study, especially to tolchlophos-methyl (I,, = 0.3 &ml). No lipid peroxidation occurred in the fungal microsomes when treated with the fungicides. In fact, no peroxidation was observed when the fungal microsomes were treated with a potent oxidation system of ascorbate iron. This may be explained by the lack of highly polyunsaturated fatty acids in this fungus. Whereas paraquat caused the uncoupling of electron flow in Cr. maydis microsomes as demonstrated by NADPH oxidation and O2 consumption, no effect was observed upon treatment with the fungicides. These compounds also did not inhibit NADPH-cytochrome P450 reductase activity. These results suggest that lipid peroxidation as the primary mode of action of these compounds is unlikely in this organism. 0 1992 Academic Press, Inc

INTRODUCTION

fungicides show effects such as inhibition of nucleic acid synthesis (IO), inhibition of respiration (1 l), thickening of cell walls (12), inhibition of nuclear division (13), mutagenesis (14), inhibition of protein synthesis (15), and lysis of mitochondria (16). However, it is not known whether the fungal responses described above reflect primary or secondary effects of the fungicides. A recent proposal put forth by Lyr and co-workers (1, 2) suggests that the basis for toxicity of dicarboximide and aromatic hydrocarbon fungicides may involve active oxygen species, as has been shown with paraquat. Bus et al. (17) were the first to demonstrate microsomal electron uncoupling with paraquat. Paraquat is rapidly reduced by microsomal NADPH-cytochrome P450 reductase; reduced paraquat , in turn, rapidly reduces dioxygen, producing superoxide anion radical (superoxide) (Fig. 1). The mechanism of paraquat toxicity has been proposed to involve both the depletion of cellular reducing equivalents

The mode of action of two major groups of fungicides, the dicarboximides and the aromatic hydrocarbons, has not been clearly established despite the efforts of many researchers. These compounds have been in use for many years in control of important crop pathogens, most notably Botrytis cinerea, the causal agent of gray mold of grapes (1, 2). These two groups of compounds are presumed to have a similar mode of action because fungal strains resistant to one group exhibit cross-resistance to the other group (3, 4). Investigations of the dicarboximide mode of action show many different effects, including mitotic instability (5), somatic segregation of chromosomes (6), inhibition of DNA synthesis (7), inhibition of cell wall synthesis (8), and increased levels of free fatty acids (9). Fungi treated with various aromatic hydrocarbon ’ To whom correspondence

should be addressed. 91

0048-3575192 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

92

ORTH

ET

AL.

Lipid Peroxidation NADPH

NADP +

-

Lipid Peroxidation

FIG. 1. Electron flow of microsomal mixed-function oxidase. Normal flow of electrons is from NADPH to reductase to cytochrome P450. Alternatively, xenobiotics such as paraquat (PQ) can uncouple electron flow and receive electrons from the reductase. Upon reduction, paraquat autoxidizes to produce superoxide. Certain xenobiotics, such as perfuorobenzene (R) can bind to cytochrome P450 and cause formation of superoxide through autoxidation of the heme.

and radical damage, including lipid peroxidation, initiated from superoxide production (17). Other workers have proposed a mechanism similar to that of paraquat and other herbicides where electron flow is uncoupled from metabolic processes resulting in the reduction of dioxygen (1, 2). This proposal was based on the observation that increased microsomal NADPH oxidation and decreased activity of cytochrome c reductase occurred in incubations containing these fungicides. Since lipid peroxidation can have many consequences in the cell, this theory would also account for the many varied effects observed by previous investigators (18, 19). In this study, we further tested the hypothesis that aromatic hydrocarbon and dicarboximide fungicides are toxic to Ustilago maydis by virtue of active oxygen generation with resultant lipid peroxidation. We compare these effects to those of paraquat, which is known to interfere with the microsomal electron transport pathway. MATERIALS

AND

METHODS

Culture methods. U. maydis (DC) Cda., ATCC 14826, was cultured in liquid medium (20) supplemented with 2 g/liter yeast extract. Cultures were incubated at 30°C while agitated on a rotary shaker at 230 r-pm. Sporidia were harvested by centrifugation, washed with distilled water, and re-

suspended in defined nutrient medium 72 (21) for growth experiments. Chemicals. Thiobarbituric acid, NADPH, cytochrome c (Type VI), and ADP were obtained from Sigma Chemical Co. (St. Louis, MO). All reagents were analytical grade and were used without further purification. Pesticides. All fungicides were added to the cultures or microsomes as methanolic solutions with appropriate solvent controls. The solvent did not exceed 0.5% of the culture medium or reaction mixture. The chemicals used were gifts from the following (Fig. 2): Tolclophos-methyl (I) was from Sumitomo Chemical Co.; iprodione (II) was from Rhone-Poulenc; myclozoline (BAS436F) (III) was from Lodvica Gullino, Institute of Plant Pathology, University of Torino, Torino, Italy; chloroneb (IV) was from E. I. DuPont de Nemours, Wilmington, DE; and vinclozolin (V) was from BASF Aktiengesellschaft. Paraquat (methyl viologen) was purchased from Sigma Chemical Co. Toxicity measurements. Sporidia from log-phase U. maydis wild type cultures were standardized to a concentration of 1 x lo6 sporidia/ml in defined nutrient medium (21) containing various concentrations of one of the above inhibitors. During incubation, measurement of growth was determined by the change in optical density at

FUNGICIDES

AND

LIPID

PEROXIDATION

I. Tolclophos-methyl

FIG. 2. Structures

0

V. Vinclozolin

IV. Iprodione of the aromatic

93

CJ. maydis

II. Chloroneb

Cl

III. Myclozoline

IN

hydrocarbon

450 nm. Measurements were taken every 2.5 hr for a 12-hr incubation period. The I,, is the concentration which inhibits growth of the fungus by 50% over a lo-hr period. Preparation of microsomal fraction. Rat liver microsomes were a gift of Dr. Steven D. Aust of Utah State University. Fungal microsomes were isolated according to the procedure of Orth et al. (22), with some modifications. All solutions were kept at 4°C and were argon-purged. U. maydis sporidia were harvested and washed in 25 mM sodium phosphate buffer, pH 7.0. The sporidia were then protoplasted using lytic enzyme obtained from Trichoderma harzianum isolated according to the procedure of Waterfield and Sisler (23). Sporidia from 2 liters of a log-phase culture were harvested, resuspended in 100 ml of lytic enzyme preparation (0.3 mg/ml), and gently agitated for 1 hr at 30°C. The protoplasts were pelleted at ll,OOOg for 10 min and washed twice in 100 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA, 4 mM magnesium chloride, and 20% glycerol. The pellet was then resuspended in the above buffer containing 0.2 mM dithiothreitol and 0.2 mM methanolic phenylmethylsulfonyl fluoride (PMSF). This solution was homogenized mechanically with four slow, even strokes. The homogenate was centrifuged for 20 min at 11 ,OOOgat 4°C. The postmitochondrial supernatant was filtered though glass wool to

and dicarboximide

fungicides

used

in this

study.

remove the floating lipid and then centrifuged for 90 min at 150,OOOg at 4°C. The microsomal pellet was resuspended in buffer containing 50 mM Tris-HCl, pH 8.0, and 20% glycerol using a hand-held tissue homogenizer. This microsomal fraction was then used for subsequent enzyme assays. Enzyme assays. NADPH-dependent cytochrome P450 reductase activity was assayed by monitoring the reduction in cytochrome c at 550 nm (e55,,nm = 21 rn&- ’ . cm- ‘) at 25°C. Incubations contained 0.5 mg/ml of microsomal protein, 100 ~.LMcytochrome c, and 100 $V NADPH in 300 mM sodium phosphate buffer (pH 7.0). NADPH oxidation was assayed in a 50 mM Tris-HCl buffer (pH 7.5) containing 100 p.M NADPH and 0.5 mg/ml of microsomal protein. NADPH oxidation was monitored at 340 nm (E~~,,~” = 6.2 m&l-’ . cm-‘) at 25°C. Oxygen consumption. Oxygen consumption was assayed in 50 mJ4 Tris-HCl (pH 7.0) buffer containing 100 pM NADPH and 1 mg/ml of fungal microsomal protein at 25°C. Oxygen concentration was monitored using an oxygen electrode from Yellow Springs Instrument Co. (Yellow Springs, OH). Lipidperoxidation. Microsomal lipid peroxidation reaction mixtures contained ADP-Fe3+ (1.7 mM ADP, 0.1 mM FeCl,) or ascorbate-Fe3+ (0.1 mM ascorbate, 0.1

94

ORTH ET AL.

mM FeCI,), 0.5 mg/ml rat liver or U. maydis microsomal protein, and 100 p& NADPH in 50 mM Tris-HCl buffer, pH 7.5. Reactions were initiated by the addition of NADPH. Iron chelate solutions were made by the addition of FeCl, to the appropriate chelate solutions and then the pH was adjusted. All iron chelate solutions are expressed as concentrations of FeCl,. Lipid peroxidation was assayed by malondialdehyde formation (24). Reactions were carried out in a shaking water bath under air at 37°C for rat liver microsomes and at 25°C for U. maydis microsomes. Fatty acid analysis. U. maydis sporidia (0.07 mg/ml initial dry wt) were incubated in liquid medium 72 (21) for various time intervals, then harvested by centrifugation, washed twice with distilled water, and lyophilized. Dried cells were extracted twice with chloroform:methanol(2: 1, v/v) (25,26) and the extract was prepared for gas-liquid chromatography by the following procedures. After separation of neutral from polar lipids on a chromatographic column packed with Biolsil-A (Bio-Rad Laboratories), neutral lipids were separated on Silica Gel G (E. M. Laboratories, Inc.) thin-layer chromatography plates (27). Plates were developed in hexane:diethyl ether:acetic acid (80:20:1, v/v/v). Bands were located after spraying with a 0.1% 2,7-dichlorofluorescein in 95% ethanol by viewing under longwave ultraviolet irradiation, and the fractions eluted with diethyl ether. Polar lipids and triacyl glycerols were saponifted in 80% ethanol containing potassium hydroxide (0.15 g/ml) at 60°C for 4 hr. After acidification with 9 N HCl, the fatty acids were partitioned into hexane. Methyl esters of the free fatty acids and fatty acids from the polar lipid and triacylglycerol fractions were prepared by incubation with 5 ml boron trichloride methanol for 5 min at 60°C and partitioning into hexane. Fatty acid esters were analyzed using a Varian Aerograph 2100 Series GLC equipped with a 1.8 m x 2 mm (i.d.) glass U-tube column packed with Gas Chrom P, 80/100-mesh

coated with 13% DEGS (diethyleneglycol succinate) + 0.1% H,PO, (oven temperature , 180°C). Identification and quantification were based on the relative retention time of a known amount of the methyl ester of palmitic acid as an external standard. RESULTS

A proposed consequence of superoxide production in the presence of transition metals is lipid peroxidation (28). If the fungicides studied here uncouple microsomal electron flow as previously proposed (1, 2)) one would observe an increase in NADPH oxidation, inhibition of the NADPH cytochrome P450 reductase activity, an increase in dioxygen consumption, and an increase in lipid peroxidation upon treatment of microsomes of sensitive fungi with the fungicides (Fig. 1). We initially determined the toxicity of these fungicides in U. maydis and then assayed for the above effects. For clarity, data are shown for representative compounds since results were consistent for all fungicides. Toxicity Measurements

The toxicity of selected aromatic hydrocarbon and dicarboximide fungicides was determined by inhibition of growth in liquid shaking cultures (Table 1). These data show that U. maydis is quite sensitive to these TABLE 1 Fungicide Concentration Giving 50% Inhibition of Growth in Ustilago maydis Fungicide Aromatic hydrocarbons Chloroneb Tolchlophos-methyl Dicarboximides Vinclozolin Iprodione Myclozoline a Mean tion was mined by time with

(I,,)

I,, bg/mlY 4.5 0.3 1.3 1.9 5.3

of triplicate cultures. Initial cell concentra0.4 x lo6 sporiditiml. Growth was determeasuring the change in optical density over varying concentrations of the fungicides.

FUNGICIDES

AND LIPID PEROXIDATION

fungicides. The greatest sensitivity, as indicated by the concentration which would cause 50% inhibition in growth rate, was found with tolchlophos-methyl, followed by vinclozolin and iprodione. Chloroneb and myclozoline were the least toxic to U. maydis, but were still very efficient at inhibiting growth at low concentrations. Lipid Peroxidation

To determine whether microsomal metabolism of these fungicides resulted in lipid peroxidation, we assayed for malondialdehyde formation in microsomes of U. maydis. Varying concentrations of vinclozolin were tested and no appreciable malondialdehyde content could be detected (Table 2). In fact, when ascorbate iron was used to chemically initiate lipid peroxidation, none was detected in the case of fungal microsomes. As a positive control, we used the ascorbate-Fe3+ system to peroxidize rat liver microsomes and observed high levels of malondialdehyde formation. The rate of over 8 nmol/min/ml obtained with rat TABLE Malondialdehyde Rat

2

Formation by Usiilaga Liver Microsomes”

maydis

or

Malondialdehyde (nmoVmin/mg protein) microsomes No additions Vinclozolin (64 &ml), ADP-Fe3+ ADP-Fe3+ Ascorbate-Fe3 + Rat liver microsomes No additions ADP-Fe3+ Ascorbate-Fe3+ Rat liver and U. muydis microsomes Ascorbate-Fe3+ U. maydis

NDb ND ND ND ND 1.30 0.77 0.78

a Microsomal reaction mixtures contained 0.5 mg of microsomal protein/ml, 0.1 m&f NADPH, ADP-Fe3+ (1.7 mM ADP, 0.1 rnkf FeCl,), or ascorbate-Fe3+ (0.1 m&f ascorbate, 0.1 mM FeCl,) in 50 m&f Tris-HCI, pH 7.5. Incubation and assay conditions are described under Materials and Methods. b Not detected.

IN (1. maydis

95

liver microsomes is similar to previous reports (28). We also tried to induce lipid peroxidation in fungal microsomes by the addition of ADP-Fe3+ and NADPH. ADPFe3+ has been previously demonstrated to be a potent initiator of microsomal NADPH-dependent lipid peroxidation (28). Again, no malondialdehyde was observed in fungal reaction mixtures. High levels of malondialdehyde were observed in reactions containing rat liver microsomes (Table 2). The absence of malondialdehyde formation in fungal microsomes could be due to the presence of high levels of antioxidants which would very effectively inhibit lipid peroxidation. This possibility was tested by adding fungal microsomes to rat liver microsomes in the presence of NADPH and ADP-Fe3 + and assaying for lipid peroxidation. The rate of lipid peroxidation of rat liver microsomes was not affected by the presence of fungal microsomes (Table 2). Fatty Acid Analysis

It is well known that highly polyunsaturated fatty acids are most susceptible to lipid peroxidation (28). Malondialdehyde is a minor product of lipid peroxidation and is formed only from fatty acids with three or more double bonds (28). It is thus possible that, due to the fatty acid composition of these fungal microsomes, they would not yield malondialdehyde as a major product. Therefore, we measured the amount of each type of fatty acid present in 17. maydis to determine whether a high degree of saturation existed among the fatty acids present in this fungus. Table 3 shows that the predominant fatty acids are linoleic acid (18:2; 46%), palmitic acid (16:O; 35%), and oleic acid (l&l, 18%), with only trace amounts of linolenic acid (18:3). No fatty acids with a higher degree of saturation were detected in this organism. Microsomal

Electron

Transport

Lipid peroxidation is only one of many consequences of free radical damage. The

ORTH

96

ET

TABLE Fatty

Acid

Analysis

of Ustilago

AL.

3

maydis

Sporidia

Grown

for

8 hr

Fatty acids (ug/mg dry wt)” Lipid fraction

16:0

16:l

18:O

18:l

18:2

18:3

Total

Phospholipid Triacylglycerol Free fatty acids Total

3.9 13.0 0.7 17.6

0.4 0.2 TRC 0.6

0.1 0.5 0.1 0.7

1.4 7.6 0.1 9.1

15.3 7.6 0.1 23.0

TRCb TRC ND TRC

20.7 28.8 1.0 50.5

u Mean of dunhcate flasks from two exneriments; initial cell concentration detected as described under Materials and Methods. b TRC, trace amount detected; ND, not detected.

absence of polyunsaturated fatty acids and malondialdehyde formation in fungal microsomes does not negate the possibility of electron uncoupling and superoxide formation in fungal microsomes. If these fungicides were reduced by NADPH-cytochrome P450 reductase, as is the case with paraquat, they should competitively inhibit the reduction of cytochrome c by the reductase. However, treatment of U. may&s microsomes with vinclozolin or tolchlophosmethyl gave no inhibition of the reductasecatalyzed cytochrome c reduction (Table 4).

The oxidation of NADPH in U. maydis microsomes upon incubation with vinclozolin was compared to that observed with paraquat in the same system. It is clear that increasing concentrations of paraquat resulted in increasing NADPH oxidation (Fig. 3). In the case of vinclozolin, however, no stimulation of NADPH oxidation TABLE Effect

of Fungicides Ustilago

Fungicide Control Vinclozolin (64 mg/ml) Control Tolchlophos-methyl (6.4 mg/ml)

was observed. The rate of NADPH oxidation remained the same despite increasing the concentration of fungicide to 50-fold its I,,. Dioxygen

Consumption

The possibility remained that alternate pathways existed for the reduction of these fungicides, such as by cytochrome P450. Regardless of the path of electron flow, electron uncoupling and superoxide formation necessitate the consumption of dioxygen. A clear increase in NADPH-dependent dioxygen consumption was observed upon incubation of U. maydis microsomes with increasing concentrations of paraquat up to 30 kg/ml (Fig. 4). However, this stimulation was not observed when the fungal

* __-___. ,N” ,,.).

4

on Cytochrome c Reduction maydis Microsomes

was 0.09 mgiml. Fatty acids were

in

Reduced cytochrome c (nmol/min/mg protein)” 19.0 2 0.1 21.0 * 0.4 17.3 2 0.2 17.2 -r- 0.3

0 Mean of triplicate experiments. Reduced cytochrome c was measured as described under Materials and Methods.

0

I

4

20 40 60 80 Inhibitor concentration, pg/ml

100

FIG. 3. NADPH oxidation by II. maydis microsomes incubated with increasing concentrations of vinclozolin (0) and paraquat (0). NADPH oxidation was assayed in a 50 mM Tris-HCI buffer @H 7.5) containing 100 PM NADPH and 1 .O mglml U. maydis microsomes as the enzyme source. Change in absorbance as NADPH was oxidized was measured at 340 nm. Values are means of triplicate experiments + standard deviation.

FUNGICIDES

AND

LIPID

0 1 0

PEROXIDATION

I 20

40 Inhibitor

FIG. 4. Oxygen crosomes incubated vinclozolin (0) and tion was assayed in containing 100 PM crosomal protein at cate experiments 2

60 80 concentration,

100 pg/ml

120

140

consumption by U. maydis tniwith increasing concentrations of paraquat (0). Oxygen consump50 mM Tris-HCI (PH 7.0) buffer NADPH and 1 mglml fungal mi25°C. Values are means of triplistandard deviation.

microsomes were incubated with vinclozolin up to concentrations loo-fold higher than its EDso (1.3 kg/ml). DISCUSSION

Even though several fungicides are known to interact with electron transport pathways, the possibility that toxicity of these fungicides is due to oxygen free radicals is only beginning to receive attention (1, 2, 29, 30). Other pesticides, such as paraquat, are known to generate free radicals which may result in the peroxidation of cellular lipids (17). Lipid peroxidation has also been proposed to be involved in the toxicity of other compounds, such as the N-phenyl imide herbicide S-23142 (31), the diphenyl ether herbicides acifluorofen and oxyfluorofen (32), and the nitrodiphenyl ether herbicides (33). In addition, lipid peroxidation may be involved as a secondary mode of action of the sterol biosynthesis inhibitors (34). In this study, we have attempted to determine the role of lipid peroxidation in the mode of action of several aromatic hydrocarbon and dicarboximide fungicides in the highly sensitive organism U. maydis. Previous investigators had proposed that these compounds initiated lipid peroxidation by

IN

I/. maydis

97

interfering with microsomal electron transport (1, 2). We found no evidence for inhibition of microsomal electron transport or for microsomal lipid peroxidation. These fungicides neither inhibit NADPHcytochrome P450 reductase activity as measured by cytochrome c reduction nor stimulate microsomal NADPH oxidation. The former observation indicates that these fungicides are not reduced by the reductase, whereas the latter observation indicates that they are not reduced by microsoma1 electron transport. If these compounds were acting as redox cycling agents, similar to paraquat, one would expect an increase in consumption of NADPH and dioxygen. In the case of paraquat, this was verified in our study. However, in the case of the fungicides, no increase in NADPH oxidation or dioxygen consumption was evident. Concentrations up to loo-fold higher than those needed to inhibit growth were tested. Since one would expect the proposed target site to be as sensitive as growth is to the fungicide, we conclude that these compounds probably do not act by producing oxygen radicals as their primary mode of cytotoxicity in Ustilago

maydis.

We confirmed previous work showing that treatment of rat liver microsomes with NADPH and ADP-Fe3 + or with ascorbate and Fe3+ readily caused lipid peroxidation (28). However, these agents were not able to initiate peroxidation of fungal microsomes. Further examination revealed that the lack of peroxidation was not due to the presence of inhibitory factors, such as antioxidants, but rather due to low levels of polyunsaturated fatty acids (PUFA) in U. maydis microsomes. Investigators have shown that lower PUFA content lowers the rate of lipid peroxidation (28, 35). For example, Tien and Aust (28) found that rabbit liver microsomes, which have a lower PUFA content than rat liver microsomes, do not catalyze lipid peroxidation as readily (36-38). Thus, lipid peroxidation would be more important and more likely in a cell

98

ORTH ET AL.

type which contained a large amount of PUFAs. In U. maydis, as in most higher fungi (39), the predominant fatty acids are linoleic (18:2), palmitic (16:0), and oleic (18:1), with only trace amounts of linolenic (18:3) acid. As shown by Tien and Aust (28), these fatty acids are not readily consumed during microsomal lipid peroxidation. Furthermore, formation of malondialdehyde requires a high content of fatty acids with three or more double bonds. Our study is in contrast with those of Lyr and co-workers, who observed an increase in malondialdehyde following treatment of Mucor mucedo with chloroneb or PCNB (2) or following treatment of B. cinerea with vinclozolin (1). Both a-tocopherol and piperonylbutoxide decreased toxicity and the level of malondialdehyde in treated cells. This difference in results may be explained by the difference in fatty acid composition between V. maydis and B. cinerea. Since B. cinerea has a much higher proportion of linolenic acid (18:3 ; 42%) compared to most other higher fungi (generally ~10%) (39), lipid peroxidation may play a more important role in this organism than in U. maydis. The observed inhibition of NADPHcytochrome P450 reductase activity in microsomal fractions of B. cinerea by vinclozolin is more difficult to explain in light of our results (1). However, inhibition of cytochrome c reduction was previously observed at fungicide concentrations over lofold greater than the I,,. This would indicate that inhibition of microsomal electron transport may be a secondary target of action, since the primary target should be sensitive at concentrations close to the I,, concentration. In addition, the possibility exists that these fungicides could trigger superoxide generation by binding to cytochrome ~450 and causing lipid peroxidation in fungi like B. cinerea. However, this appears unlikely because we detected no increase in oxygen consumption in microsomal incubations containing these fungicides . Results from this study do not support

the role of lipid peroxidation as the primary mode of action of the dicarboximide or the aromatic hydrocarbon fungicides in U. maydis. However, our results do not preclude radical damage by active species other than lipid peroxides, or free radical damage to other subcellular organelles. Further research is in progress using mutants resistant to these compounds to possibly identify their target sites and the basis for this resistance. ACKNOWLEDGMENTS

This work was supported in part by a Pennsylvania State University Intercollege Research Grant (Project 3138). Ann Orth is a postdoctoral fellow supported by National Research Service Award l-F32-ES05503-01 from the National Institute of Environmental Health. Ming Tien is the recipient of Presidential Young Investigator Award DCB-8657853 from the National Science Foundation. REFERENCES

1. W. Edlich and H. L. Lyr, Mechanism of action of dicarboximide fungicides, in “Modern Selective Fungicides” (H. Lyr, Ed.), pp. 107-118, Fisher Verlag, Jena, 1987. 2. H. Lyr, Mechanism of action of aromatic hydrocarbon fungicides, in “Modem Selective Fungicides” (H. Lyr, Ed.), pp. 75-89, Fisher Verlag, Jena, 1987. 3. P. Leroux and R. Fritz, Antifungal activity of dicarboximides and aromatic hydrocarbons and resistance to these fungicides, in “Mode of Action of Antifungal Agents” (A. J. P. Trinci and J. F. Ryley, Eds.), pp. 207-237, Cambridge Univ. Press, London, 1984. 4. P. Leroux, R. Fritz, and M. Gredt, Cross resistance between 3,5-dichlorophenyl cyclic imide fungicides and various aromatic hydrocarbons, in “Systemische Fungizide und Antifugale Verbingdungen” (H. Lyr and C. Puolter, Eds.), pp. 79-88, Akademie-Verlag, Berlin, 1983. 5. S. G. Georgolpoulos, M. Sarris, and B. Ziogas, Mitotic instability in Aspergillus nidulans caused by the fungicides iprodione, procymidone and vinclozolin, Pestic. Sci. 10, 389 (1979). 6. P. Leroux, M. Gredt, and R. Fritz, Etudes en laboratoire de souches de quelques champions phytopathogenes resistantes a la dichlozoline, a la dicyclidine, a l’iprodione, a la vinclozolin et a divers fondicides aromatiques, Med. Fat. Landbouw, Rikjsuniv. Gent 43, 881 (1978). 7. Y. Hisada and Y. Kawase, Morphological studies

FUNGICIDES

8. 9.

10. 11.

12.

13.

14.

15. 16. 17.

18.

19. 20.

21.

AND

LIPID

on antifungal action of N-(3’,5’-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximide on Botrytis cinerea, Ann. Phytopathol. sot. Jpn. 43, 151 (1977). G. Albert, Spharoblastenbildung bei Botrytis cinerea, hervorgerufen durch vinclozolin, Z. Pflanzenkrankh. Pflanzenschutz 88, 337 (1981). A. C. Pappas and D. H. Fisher, A comparison of the mechanism of action of vinclozolin, procymiodne, iprodione and prochloraz against Botrytis cinerea. Pestic. Sci. 10, 239 (1979). W. K. Hock and H. D. Sisler, Specificity and mechanism of antifungal action of chloroneb, Phytoparhology 59, 627 (1969). H. R. Kataria and R. K. Grover, Effect of chloroneb (1,4-dichloro-2,5-dimethoxybenzene) and pentachloronitrobenzene on metabolic activities of Rhizoctonia solani Kuhn., Znd. J. Exp. Biol. 13, 281 (1975). H. Lyr and G. Casperson, Anomalous cell wall synthesis in (L.) Fres. induced by some fungicides and other compounds related to the problem of dimorphism, Z. Allg. Mikrobiol. 22, 245 (1982). Kato, T. Mode of antifungal action of a new fungicide, tolclophos-methyl, in “Pesticide Chemistry” (J. Miyamoto and P. C. Keamy, Eds.), Vol. 3, pp 153-157, Pergamon Press, Oxford, 1983. S. G. Georgopoulos, A. Kappas, and A. C. Hastie, Induced sectoring in diploid Aspergillus niduluns as a criterion for fungitoxicity by interference with hereditary processes, Phytopathology 66, 217 (1976). R. W. Tillman, and H. D. Sisler, Effect of chloroneb on the growth metabolism of Ustilago muydis, Phytopathology 63, 219 (1973). G. Casperson and H. Lyr, Wirking von terrazol auf die ultrastruktur von Mucor mucedo, Z. Allg. Mikrobiol. 22, 219 (1975). J. S. Bus, S. D. Aust, and J. E. Gibson, Superoxide- and singlet oxygen-catalyzed lipid peroxidation as a possible mechanism for paraquat (methyl viologen) toxicity, Biochem. Biophys. Res. Commun. 58, 749 (1974). H. E. May and P. B. McCay, Reduced triphosphopyridine nucleotide oxidase-catalyzed alterations of membrane phospholipids. I. Nature of the lipid alterations, J. Biol. Chem. 243, 2288 (1968). W. T. Roubal and A. L. Tappel, Damage to proteins, enzymes and amino acids by peroxidizing lipids, Arch. Biochem. Biophys. 113, 5 (1966). B. W. Coursen and H. D. Sisler, Effect of the antibiotic, cycloheximide, on the metabolism and growth of Saccharomyces pastorianus, Amer. J. Bor. 47, 541 (1960). N. N. Ragsdale and H. D. Sisler, Inhibition of er-

PEROXIDATION

22.

23. 24.

25.

26. 27. 28.

29.

30.

31.

32.

33.

34.

35.

IN

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