Effects of the herbicides benzoylpropethyl and flampropisopropyl on rat liver mitochondria: An alteration in membrane fluidity?

PI-STl(‘lDh

HIOCHtMISTKY

AND

PHYSIOLOGY

21, 377-384 (1984)

Effects of the Herbicides Benzoylpropethyl on Rat Liver Mitochondria: An Alteration CHRISTIAN

and Flampropisopropyl in Membrane Fluidity?

GAUVRIT

Received August 15. 1983: accepted November 15. 1983 The action of the herbicides benzoylpropethyl and flampropisopropyl. and the corresponding unesterified acids was studied in rat liver mitochondria. The herbicides were found to (a) inhibit the mitochondrial electron transfer in complex III or at the level of ubiquinone (I,,, of 4 nmol mg protein’ for flampropisopropyl and 18 for benzoylpropethyl with succinate as a substrate); thl have an additional (however less sensitive) site of inhibition near succinate dehydrogenase: and (cl interfere with energy transfer. Sensitivity was increased 2- (benzoylpropethyl) and 3.5-fold (flampropisopropyl) as the rats age increased from 12- 13 weeks to 23-26 weeks. The free acids were far less effective. Since the herbicides benzoylpropethyl and flampropisopropyl are readily hydrolyzed by animals and since the free acids are less effective than the herbicides. it explain\ why these potentially harmful compounds have a low acute toxicity, Swelling studies in isoosmotic salts suggested that the two herbicides decrease membrane fluidity. an action which was assumed to be responsible for the electron transfer inhibition and. via inhibition of phosphate transport. the interference with energy transfer. INTRODUCTION

The herbicides benzoylpropethyl (ethyl N - benzoy1 - N - (3,4 - dichlarophenyl) - DL alaninate: BPE)’ and ( - )flampropisopropyl (isopropyl - N - benzoyl - N - (3 - chloro, 4 fluorophenyl) - L - alaninate; FIP) have been used for several years, mainly to control wild oats (Awna fatua L. and A. sterilis L.) in wheat crops (1. 2). Little is known about their biochemical mode of action in plants. The biologically active forms are thought to be the free acids derived from the herbicides: N - benzoyl -N- (3.4 - dichlorophenyl) - DL - alaninate (BP) and N - benzoyl ’ Abbreviations used: BP, benzoylprop: BPE. benroylpropethyl: CCCP, carbonylcyanide-m-chlorophenylhydrazone: FCCP. carbonylcyanide-4-trifluoromethoxyphenylhydrazone; FI, flamprop. FIP, flamproptsopropyl: I,,. concentration at which an activity is 50% inhibited: Mops, 4-morpholinepropanesulfonate; NEM. N-ethyl maleimide; PMS, phenazine methosulfate; P/O ratio, ratio between the amount of phosphate esterified to ATP and the amount of oxygen consumed in the process; SD, standard deviation; TMPD, N,N.N’,N’-tetramethyl-p-phenylenediamine; SDS, sodium dodecyl sulfate.

N - (3 - chloro,4 - fluoraphenyl) - L - alaninatc (FI) (2, 3). They display a very low toxicity towards mammals, as exemplified by the values ot their acute oral LDSo for rats: BPE, 1555 and FIP. 4000 mg kg-’ (4). Nevertheless. we found that these chemicals had a strong activity in vitro against rat liver mitochondria: they inhibited electron transfer at low concentrations and interfered with energy transfer. In this study we attempted to cxplain the mechanism of action of the two herbicides on rat liver mitochondria.

MATERIAL

AND METHODS

Mitochondria were prepared from Wistar rat livers using a standard procedure (5). Preparations were used only if they exhibited respiratory control ratios above 3 with succinate as a substrate. No endogenous respiration was observed. Respiratory studies. They were carried out at 35°C with a Clark-type oxygen electrode. When necessary, the pH variations

378

CHRISTIAN

in the medium were monitored to follow the phosphorylation of ADP. The respiratory medium was 0.3 M mannitol, 10 mM KCl, 5 mM MgCl,, 10 mM potassium phosphate, pH 7.2. Substrate concentrations were 5 mM succinate, 20 mM malate, and 10 mM glutamate. State 4 and state 3 were defined according to (6). Effects on state 3 respiration were measured by addition of the chemical under study approximately 1 min after introduction of excess ADP (1.25 mM) and the establishment of linearity. Localization of the inhibition site. Succinate-PMS oxidoreductase activity was measured polarographically in the basic reaction mixture with 5 mM succinate, 1 mM ADP, 1 mM PMS, 0.1 FM antimycin A, and 1 mM KCN. Malate/glutamatePMS oxidoreductase activity was measured similarily with 20 mM malate, 10 rrG’t4 glutamate, 1 mM ADP, 0.1 pM Antimycin A, 1 mM KCN, and 0.1 mM PMS. Cytochrome oxidase activity was assayed polarographically in the basic reaction mixture with 0.1 mM TMPD, 1 mM sodium ascorbate, and 0.1 FM antimycin A. Succinate or malate/glutamate-ferricyanide oxidoreductase activities were measured in the basic reaction mixture with the addition of 1 mM KCN and 1 mM potassium ferricyanide. Ferricyanide reduction was measured as a decrease in absorbance at 420 nm. Mitochondrial swelling. This was measured as an absorbance decrease at 520 nm. The 2-ml reaction mixtures contained 0.20.25 mg protein ml-’ and had an initial absorbance of 0.6-0.7. To determine the inhibition of CCCP-induced swelling by the herbicides, mitochondria were suspended in 150 mM NH,NO,, 10 mM Mops, pH 7.2. The herbicide under study was added approximately 1 min before the addition of 1 p,M CCCP. Rates of swelling were calculated from the initial linear phase of absorbance decrease. To determine the inhibition of valinomycin-induced swelling by the herbicides, mitochondria were suspended in 150 mM KNO,, 10 mM Mops, pH 7.2, and

GAUVRIT

the same procedure as above was followed except that 0.5 pM valinomycin was used instead of CCCP. To determine the inhibition of mitochondrial spontaneous swelling in 150 mM ammonium phosphate, 10 mM Mops, pH 7.2, mitochondria were suspended in the medium with the herbicide under study and the decrease in absorbance was monitored. To determine the inhibition of swelling by NEM, mitochondria were preincubated at 25°C for 5 min, with NEM (2 kmol mg protein-‘) then added to the indicated reaction mixture. ATPase activity. ATPase (EC 3.6.1.3) activity was measured in mitochondria (1.5 mg protein) suspended in 0.25 M sucrose, 10 mM Mops, pH 7.4. Further additions were 100 FM herbicide (67 nmol mg protein- ‘> and 3 pg oligomycin mg protein-l. The reaction was started by addition of 2.5 mM ATP-Mg and continued for 15 min. The final volume was 1 ml. The liberated phosphate was assayed by stopping the reaction with 1 ml of a freshly prepared solution containing 0.75 N H,SO,, 0.75% (NH&Mo~O~~. 7H,O, 3% Fe SO4. 7H,O, and 4% SDS. The samples were read at 740 nm 10 min after reagent addition (7). Chemicals. The DL forms of BPE and BP, and the L isomers of FIP and FI were used. The herbicides (more than 99% purity) were kindly supplied by Shell Science Laboratory, Sittingbourne, U.K. All reagents were of analytical grade. The results presented here are the means of at least three independent experiments. RESULTS

Inhibition

AND

of Electron

DISCUSSION

Flow

Table 1 and Fig. 1 show that BPE and FIP inhibited state 3 oxygen consumption with both succinate and malate/glutamate as substrates. Succinate-linked respiration was roughly twice as sensitive as malate/ glutamate-linked respiration. FIP was more potent than BPE. Even at higher concentrations the herbicides did not fully inhibit

BEN%OYLPROPETHYL/FLAMPROPISOPROPYL TABLE Inhihifion

-

of Mitochondrial Iso V&es

Herbicide

Succinate

BPE FIP BP Fl

39 (6) 14 (1.5) 300 (40) 440 (10)

1 Electron

Transjkr:

Malateiglutamate 59 (3)” 27 (5)

Nr>rc>.Values of I,, concentrations are in nmol mg ’ protein: 0.3-0.5 mg protein ml-’ was present: ADP was in excess (1.25 mM). Rats were aged 12-13 weeks. Figures in brackets are the standard deviations. (’ Only two experiments.

O2 consumption; the residual respiration amounted to 5 to 15% of the initial one and was fully inhibited by 1 mM KCN. I,, values were dependent on mitochondrial protein concentration; in the concentration range used in this study, Ij0 values expressed in micromolar concentrations were proportional to protein concentration. BP and Fl (the free acids corresponding to BPE and FIP) were inhibitors at higher (roughly one order of magnitude) concentrations. This could be due to the decrease in lipophilicity brought about by the hydro-

FIG. I. Typical experiment shovt,ing inhibition of state 3 oxygen consamption in rat liver mitochondria treated with BPE or FIP. Mitochondrial protein was 0.49 mg ml-‘. Substrate was either 5 mM succinate or 20 mM malatell0 mM glutamate. Excess ADP nsas present. Cwltrol o.rygen consumptions were 360 nmol 0? min ’ mg protein ’ ,for succinate and 220 protein f& malatel~lutrtn~trte. Rats were aged 13 wseeks. l , BPE. sawinate: c, BPE. malateiglutumate; H. FIP, vrtc~cinate; arld C, FIP. malatel~lrrtamate.

EFFECTS

ON MITOCHONDRIA

3’9

lysis of the herbicides: the concentration of, say. BP in the mitochondrial membrane was very probably lower than that of BPE. resulting in a decreased activity. We found a similar situation with the closely related herbicides diuron and neburon. The rcplacement of a methyl moeity by a n-butyl one increased lipophilicity (partition coefficient between n-octanol and water increased from 375 to 23501, and gave the molecule uncoupling properties: diuron had no effect at 1 mM whereas neburon t‘ully uncoupled potato mitochondria at 0. I m:24 (8,

9).

None of BPE, FIP. BP, or FI had any uncoupling activity as detected by state 3 respiration stimulation. The slight effect of BP and Fl explain5 why BPE and FIP are not toxic for rat\. Mammals efficiently hydrolyze them into BP and FI (IO). Thus, the potentially toxrc BPE and FIP are never present at high concentrations in the body. A similar cast: was found with thionins. which are polypeptides from cereal endosperm. They increase membrane permeability in cultured mammalian cells but are harmless irr \,ilvj kc to a very efficient degradation in the mmma1 (II). Comparison between results in Tables 1 and 3 shows that the sensitivity of rat lrver mitochondria to FIP and BPE increased with rats age. The increase wah Z-fold for BPE and 3.5-fold for FIP. An explanatio!? for this fact will be proposed later.

Table 2 shows that cytochrome oxidasc was affected neither by BPE nor FIP. l‘his was also the case for malateiglutamu~e PMS oxidoreductase activity. Succinate-PMS oxidoreductase activity was significantly inhibited; 22% (FIP) and 29% (BPE) at herbicide concentrations more than ten times higher than the I,, for succinatedriven oxygen consumption. Converselq. malate/glutamate-ferricyanide and succinate-ferricyanide oxidoreductaqe activiries were fully inhibited.

380

CHRISTIAN

GAUVRIT I: Succinate

TABLE 2 Action of FIP and BPE on Reactions Involving only Parts of the Electron Transfer Chain Reaction

FIP

Ascorbate/TMPD-0, Malate/glutamate-PMS Succinate-PMS Malate/glutamateferricyanide Succinate-ferricyanide

109 (8)O 95 (SY 78 (8.5)b 6 (lO)c 5 (5Y

2: ADP

BPE 102 (6)* 80 (15)” 71 (9S)b 100

3 (5Y 9 (8)’

Note. Results are expressed as a percentage of control value. Mitochondrial protein was 0.4-0.5 mg ml-‘; herbicide concentration was 100 yM (200-400 nmol herbicide mg protein-t). Rats were aged 19-22 weeks. Figures in brackets are the standard deviations. 0 Nonsignificantly different from 100 (P = 0.05). b Significantly different from 100 (P = 0.05). c Nonsigniticantly different from 0 (P = 0.05).

The results show some scattering; it was due to nonlinearity of the reactions which diminished accuracy in the measurement of herbicide action. These results show that the inhibition site of BPE and FIP is located between the compound donating electrons to PMS and cytochrome c. Since there is inhibition of both succinateand malate/glutamatedriven oxygen consumptions, the site is very probably located in complex II (ubiquinone-cytochrome c oxidoreductase) or at the level of ubiquinone itself. In addition succinate-PMS oxidoreductase was significantly inhibited by BPE and FIP (though at high concentrations). It could account for the greater sensitivity of succinate oxidation as shown in Table 1. Interference

I

with Energy Transfer

Inhibition release of the mitochondrial electron transfer by an uncoupler (for example, FCCP) indicates that the inhibition is due to interference with energy transfer. Figure 2 shows that BPE inhibition could be partially released by 1 $V FCCP. Similar results were found with FIP (data not shown). Table 3 gives a quantitative account of this property. (a) I,, values for in-

nmoles 02

I

I min -

\

FIG. 2. Partial release of BPE inhibition by an uncoupler (FCCP). Mitochondrial protein was 0.33 mg ml-‘. Numbers on the trace are oxygen consumption (nmol 0, mitt-’ mg protein-‘). Additions were 1, 5 mM succinate; 2, 1.25 mM ADP; 3. 100 PM BPE; and 4. I JLM FCCP.

hibition were dependent on whether ADP was limiting or in excess. It means they depended on phosphate potential. (b) I,, values found in the presence of excess ADP greatly differed from those found with uncoupled mitochondria. (c) P/O ratios were affected by BPE and FIP, this would not occur if their action were strictly limited to electron transfer inhibition. These results denote an interference with energy transfer. Hence, BPE and FIP display a multi-site action in rat liver mitochondria; (a) inhibition of the electron transfer in complex III (ubiquinone-cytochrome c oxidoreductase) or on ubiquinone; (b) inhibition of succinate-PMS oxidoreductase; and (c) interference with energy transfer. The latter action could be achieved three ways, (a) inhibition of ATPase, (b) inhibition of phosphate transport, or (c) inhibition of ATP-ADP exchange. To see whether BPE and FIP interfered with energy transfer in a similar way to oligomycin, their action on ATPase activity was studied. Action on ATPase Activity Control ATPase activities were 50-60 nmol hydrolyzed ATP min- 1 mg protein-‘. The herbicide concentrations we used (67

RENZOYLPROPETHYL/FLAMPROPISOPROPYL TABLE

EFFECTS

ON

381

MITOCHONDRIA

3

Is,, Values for Several Mitochondrial Activities Affcted by BPE and FIP I,,, (nmol herbicide

mg protein-l) BPE

Herbitide

state 3 IADP limiting)

state 3 (ADP in excess)

Uncoupled state

P/O 1 ratio

BPE FIP

47 (2) 7 (2)

18 (61 4 (I)

109 (3) 35 (III

68 (5) 16 (3)

Note. Mitochondrial protein was 0.3-0.5 mg ml-l; sub\trate was 5 rnM succinate; excess ADP was 1.25 mM; limiting ADP was 0.125 mM. Uncoupled state was obtained by addition of 1 pM FCCP to mitochondria oxidizing succinate. P/O ratios in the control experiments were 1.5- 1.7. Rats were aged 23-26 weeks. Figures in brackets are the standard deviations.

nmol mg protein-‘) brought about a 80% inhibition of electron transfer in respiratory studies. In the presence of 3 p,g oligomycin mg protein ~’ ATPase activity was reduced to 36% of control value (SD = 12%). In the presence of BPE and FIP it was, respectively, 101% (SD = 2%) and 102% (SD = 2%). The conclusion is that neither BPE nor FIP inhibited ATPase activity. Hence, we studied their effect on phosphate transport. Action on Phosphate

Transport

After suspension in isoosmotic ammonium phosphate, mitochondria swell. This is interpreted as phosphate entry into the mitochondria through exchange with hydroxide ions. Their exit counterbalances the production of H+ ions outside the mitochondria and the appearance of hydroxide ions inside the mitochondria, due to the entry of NH, (outside the mitochondria, NH, + H+ originate from NH,+ and, after NH, permeation through the mitochondrial membrane, NH, recombines with H ’ inside the mitochondria, yielding NH4+ and hydroxide ion). The resulting ammonium phosphate entry leads to mitochondrial swelling. In agreement with this interpretation, the phosphate transport inhibitor NEM strongly inhibited this swelling (Fig. 3). So did, but to a lesser extent, BPE and FIP.

I

0.’ A520

1 min

FIG. 3. Spontaneous sbvelling of mitochondricr SII.\ pended in 0.15 M ammonium phosphate. Inhibition hs, 100 )*M each of BPE, FIP. BP. und FI. und 2 PIHI)/ mg protein - ’ NEM. The results presented hew c~mw from one experiment out of’ three n~ifh .similar rc..5ulf.\

BP and FI had no effect, even at high con-centration (1 mM). We can thus infer that interference of BPE and FIP in energ] transfer results from the partial inhibition of phosphate transport into the mitochondrion. These herbicides could be specific inhib,itors of the phosphate transporter. An alternative explanation could be that they diminish membrane fluidity and consequently inhibit all transports dependent on it. Such action was demonstrated for dinitroaniline and carbamate herbicides as well as for perfluidone and dibromothymoquinone ( I?-16). Hence, we studied the effect of BPE and FIP on mitochondrial swelling in various salts. Inhibition qf S~~*elling in NH4N07 und KNO, Valinomycin-induced swelling in KNO, is the result of the entry into the mitochondria of nitrate and potassium ions (the latter via the complex valinomycin-K+), down the KNO, chemical potential gradient. Gramicidin-induced swelling in KNO, is similarly explained, except that, unlike valinomycin. gramicidin does not form permeant complexes with K.‘. but instead forms pores in the membrane, whose diameter allows K+ to go through. CCCPinduced swelling in NH,NO, is the result of the entry of nitrate and NH, into the mi-

382

CHRISTIAN

no FIP

‘.’

no v

A520

I

1 min

I

4

FIG. 4. Inhibition of valinomycin-induced swelling in isoosmotic KNO, by FIP. Additions were FIP, micromolar concentrations as indicated on the drawing; valinomycin (V). 0.5 FM. The results presented here come from one experiment out of three with similar results.

tochondria. As CCCP permeabilizes the mitochondrial membrane to H+ ions, the dissociation of NH,+ into NH, and Hf outside the mitochondria and the association of NH, and H+ inside the mitochondria are not inhibited by the ApH that would built up if the movements of H+ ions through the membrane were restricted. In these studies, nitrate salts were chosen instead of chlorides since nitrate is a far better permeant ion than chloride (17). Using nitrate salts allows working in conditions in which the anion permeation is not the limiting factor in the swelling; this is not always true with chloride salts. With nitrate salts only good mitochondria can be used since the smallest cation permeability is expressed as a swelling. The mitochondria used in this study were satisfactory since they did not swell in 0.15 M KNO,, and swelled only very slowly in 0.15 M NH,NO,. Figure 4 shows a typical result obtained with KNO, swelling. ISo values for valinomycin-induced swelling were 50 nmol mg protein r (BPE) and 71 nmol mg protein-t (FIP). Gramicidin-induced swelling was affected to the same extent by both herbicides (data not shown). Conversely, BPE

GAUVRIT

and FIP had little or no effect on CCCPinduced swelling. BP and FI triggered a swelling in mitochondria suspended in both salts and accelerated valinomycin-, gramicidin-, and CCCP-induced swellings (for instance, 48% (BP) and 36% (FI) at 100 pM (150 nmol mg protein-‘) for valinomycininduced swelling). This can be interpreted as a permeabilization of the mitochondrial membrane to H+ and K+ ions (which was not conspicuous in respiratory studies since neither BP nor FI accelerated state 4 rates). Whether this increase in permeability was accompanied with variations in fluidity could not be determined by means of swelling experiments. A decrease in membrane fluidity brought about by BPE and FIP can explain these results. Valinomycin is a mobile K+ ionophore and has to shuttle from one side of the membrane to the other to drive K+ inside the mitochondrion. A decrease in membrane fluidity is expected to slow down the process, resulting in the lowering of the swelling rate. The same explanation holds for swelling in NH,NO, since CCCP is a mobile H+ transporter. Gramicidin pores are formed by gramicidin dimers. Two gramicidin monomers must associate to form a pore spanning from one side of the membrane to the other one (18). It results in the concentration of gramicidin pores (dimers) depending on the probability of monomer association, which, in turn, is dependent on the mobility of gramicidin monomers in the plane of the membrane: a decrease in gramicidin-induced swelling can be attributed to a decrease in membrane fluidity. However, there is a discrepancy between the extent of the swelling inhibitions in NH,NO, (CCCP induced) and in KNO, (valinomycin induced). The reason could be the difference in the molecular weights of the two carriers; CCCP, 205 Da and valinomycin, 1111 Da. The bigger molecule could be more affected by a decrease in membrane fluidity. An alternative hypothesis could be that the two molecules solu-

BENZOYLPROPETHYL/FLAMPROPISOPROPYL

bilize in areas of the membrane differing in their location and their fluidity properties. It can be concluded that the inhibition of PO, transport by BPE and FIP is not specific to this transporter. It probably reflects a decrease in membrane fluidity whose effects can be observed on all transporters dependent on membrane fluidity for their activity. Moreover, it can be hypothesized that the inhibition of electron transfer described above is a result of a decreased membrane fluidity. Such a situation was suggested in (12- 16); the multisite action of several herbicides was explained by a decrease in membrane fluidity affecting the inhibited reactions. Such a reasoning could be held to explain the multisite action of BPE and FIP. A decrease in membrane fluidity could result in inhibitions of electron transfer (Complex III and succinate dehydrogenase) and in interference with energy transfer (via inhibition of phosphate transport). In our study the most potent inhibitory action of BPE and FIP was in the succinate-ubiquinone oxidoreductase part of the electron transfer chain. The presence of uniquinone in this part of the chain could be a reason for an action of membrane fluidity at this level since mobility is required for ubiquinone to complete its oxidoreduction cycles coupled to proton extrusion (19). The mitochondrial electron transfer has a higher sensitivity in rats aged 23-26 weeks than in rats aged 12-13 weeks (Tables 1 and 3). If BPE and FIP decrease membrane fluidity. their target could be the lipidic part of the membrane. Variations with age in the lipid composition of the mitochondrial membrane could be a reason for such differences in efficiency. Indeed, Kersnen rt ~1. (20) found significant changes in fatty acid composition of phospholipids in rat liver mitochondria. Even though they compared 0-, I-, and 4-day-old and adult rats instead of 12- to 13- and 23to 26-week-old rats as we did, this indicates that variations in lipid composition (not only phospholipids) should be taken into

EFFECTS

account served.

ON

MITOCHONDRIA

to interpret

the results

ACKNOWLEDGMENTS I am grateful to Dr M. Briquet and Professor A Goffeau for advice and discussions. The rat\ used 1~1 this study were kindly supplied by the Laboratoire de., Aliment5 de I’Homme. INRA, Dijon. France.

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GAUVRIT

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