EFFECTS OF THE HERBICIDE 2,4-DB AND

FUNGICIDE CAPTAN ON REACTIONS OF

MITOCHONDRIA AND CHLOROPLASTS
MILKA BUDIMIR, MARIJANAPLESNICARand R. KIdAJIC
Department of Pesticides INEP 11080 Zemun, Yugoslavia

The effects of the herbicide 4(2,4-dichlorophenoxy)butyric acid (2,4-DB) and fun-

gicide N-(trichloromethyltio)-4-cyclohexene-l,2-dicarboximide (captan) on electron
transport processes of mitochondria and chloroplasts have been investigated. Chlorop-
lasts, isolated from spinach leaves (Spinacia oleracea L.), were treated with pesticide
prior to the addition of electron acceptor and ADP. White potato (Solanum tuberosum
L.) mitochondria were either incubated with pesticide before the addition of substrate,
or they were treated with pesticide after the addition of substrate and ADP. Captan
inhibited oxidation of malate by mitochondria and acted as an uncoupler. With succi-
hate as sunstrate captan was found to stimulate state 4 respiration, as substrate captan
was found to stimulate state 4 respiration, with the loss of coupled phosphorylation
only at higher concentrations of fungicide. The herbicide 2,4-DB appeared to be 5 to
I0 times less effective than captain. Both compounds inhibited phosphorylation-
coupled succinate oxidation at higher concentrations and malate-coupled phosphory-
lation at lower concentrations. They acted as inhibitors of NADH-cytochrome c reduc-
tase.
Both pesticides inhibited noncyclic electron transport in chloroplasts. The rate of
ferricyanide reduction in the presence and absence of phosphorylating agents was
reduced, and although the rate of ATP generation was reduced also, the P/2e ratio was
not changed much under the influence of pesticides.

Captan and 2,4-DB have been widely applied as fungitoxic and herbicidal com-
pounds, respectively. Since the toxicity of captan has been ascribed to its affinity for
sulfhydryl groups (Siegal and Sisler 1968), several mitochondrial enzymes, as well
as the process of oxidative phosphorylation have been found sensitive to it too
(Nelson 1971).

2,4-DB is converted by beta-oxidation to the corresponding phenoxyacetic acid,

and it is considered that it exhibits herbicidal properties as such (Wain 1964).
Phenoxyacetic acid compounds have been found to affect different physiological
processes in plants including protein synthesis, respiration, and photosynthesis
(Penner and Ashton 1966). 2,4-D and related compounds were described as uncoup-
lers of oxidative phosphorylation (Wedding and Black 1962, Matlib and Kirkwood
1972) and inhibitors of the Hill reaction (Moreland and Hill 1962) and cyclic
photophosphorylation (Chkanikov et al. 1966).

Presented at the Third International Congress of Pesticide Chemistry (IUPAC), Helsinki, Finland,
1974.

Archives of Environmental Contamination 166

and ToxicologyVol. 4, 166-174 (1976)
9 1976 by Springer-Verlag New York Inc.
 Effects of 2,4-DB and Captan 167

The present investigation was carried out to examine the effects of captan and
2,4-DB on the electron transport and related energy conservation processes in
isolated mitochondria and chloroplasts.

Material and methods

Mitochondria were isolated from white potato (Solanum tuberosum L.) tubers
according to Bonner (1967). Mitochondrial inner membranes were isolated after
disruption of mitochondria by sonification treatment and separation on discontinous
sucrose gradient (Douce 1973). Oxygen uptake was measured at 20~ in a three-ml
stirred cell containing a built in Clark oxygen electrode (Delieu and Walker 1972).
The sequence of additions to the cuvette was as follows: reaction medium,
mitochondria (0.6 mg of protein), substrate and adenosine diphosphate (ADP) (0.5
/xmoles). Succinate (7 mM) and malate (33 mM) were chosen as the primary
substrates to examine succinoxidase and NAD-linked oxidase systems, respectively.
The compound being examined, or corresponding solvent (control) was introduced
into the cuvette either ten rain before the substrate (method B), or during the second
state 4 (method A) (Ikuma and Bonner 1967). The respiratory states, respiratory

Succinateoxidation
40 pNI 02/min
/JM 02/min
35' 35

30:
~ ~ 1 1 2 3O

25 25 0 9 0

20,

:J"
15'

10 10'
IVz ~
5'

9 0 9 )
o. a 11o 1.'6 i.o 216 313 0.33" 110 "1.6 2'.0 2'.6 3.3
X 10--4M X 10--4M
Captan
Fig. 1. Effects of captan on succinate oxidation. A: method I application; pesticide was
added in second state 4, after the substrate and ADP. B: method [I application; mitochondria
were preincubated with pesticide for 10 min. Mitochondria-0.6 mg protein, succinate - 7
rru'r ADP - 165 p~M. Roman numerals (III and IV) denote respiratory state 3 and 4,
subscripts (1, 2 and 3) indicate the first, second and third.
168 M. Budimir et al.

control (RC) and ADP/O ratios were defined and calculated according to Chance and
Williams (1956). The assay for determination of NADH2 cytochrome c oxidore-
ductase activity was as described by Douce et al. (1973).

Chloroplasts were isolated from spinach leaves (Spinacia oleracea L.) Laccording
to Walker (1967). Oxygen evolution and noncyclic photophosphorylation were
measured at 20~ by a Clark oxygen electrode (West and Wiskich 1968). The
sequence of additions to the cuvette was as follows: reaction medium (Walker
1967), chloroplasts (120 /zg of chlorophyll per three ml), ten /zl of examined
compound or corresponding organic solvent (control), incubation for 2 min, 2.5
/~moles of potassium ferricyanide, then light on (basic rate of O~ evolution). After
two min, 0.3/xmoles of ADP was added (ADP-stimulated rate) and upon its exhaus-
tion (ADP-limited rate), 20 /xmoles of ammonium chloride was added (NH4CI-
stimulated rate).
Results
Effects on mitochondrial respiration. Effects of captan on succinate and malate
oxidation by white potato mitochondria are shown in Figures 1 and 2. When applied
after succinate and ADP (Fig. 1A), captan inhibited the state 3 respiration and
stimulated state 4 respiration. At 3 x 10-4/14 captan, the inhibition of state 3 was
47%, the stimulation of state 4 was 33%. When incubated with mitochondria before
the addition of succinate and ADP (Fig. IB), 3 x 10-4M captan induced 50%

Malate oxidation
02/min
/~1 02/min

30 30

25 25,

20 20,

15 15

10 10
111

5
IV3 ~ 5
IV 1 0 9

0 9 .0 II

0.33 lio 1.6 2.o 216 3.a 0133 1.0 1[6 2.0 2.6 3~3
X 10--4M X 1"0- 4 M
Captan
Fig. 2. Effect of captan on malate oxidation. A: method I application. B: method II
application. Mitochondria - 0.6 mg protein, malate 30 mM, ADP - 165 p.M.
 Effects of 2,4-DB and Captan 169

inhibition of state 3. At lower concentrations captan stimulated state 4 respiration,

and inhibited it at higher concentrations.

Captan influenced malate oxidation (Fig. 2) by inhibiting both state 3 and 4

respiration. Fifty percent inhibition of state 3 and state 4 respiration was obtained at
3 x 10-4M, respectively. When incubated with mitochondria before the addition of
malate and ADP (Fig. 2B), the lowest applied captan concentration, 3 x 10-SM,
induced 60 % inhibition of state 3 respiration. The inhibition of state 4 respiration
was much less pronounced.

Herbicide 2,4-DB inhibited state 3 respiration and stimulated state 4 respiration

when applied after succinate and ADP (Fig. 3A). Maximal inhibition of state 3
respiration (40%) and stimulation of state 4 respiration (11%) was induced by 1.6 •
10"ZM 2,4-DB. When incubated with mitochondria before the addition of succinate
and ADP (Fig. 3B), 2,4-DB at 1.6 x 10"aM induced 69% inhibition of state 3
respiration. State 4 respiration was stimulated up to 37% at 6 x 10-4M 2,4-DB and
then inhibited.

2,4-DB inhibited both state 3 and state 4 respiration when applied after malate

Succinate oxidation

~/JM 02/min

25- ~ 1112 25'

20 'v3 20. I1

15 15~

10

5-
iV 1 i

0 1.0:3.3 6.6 10 16 ) 1.0 3.3 6.6 1'0 16

x 10--4M X 10--4M

2,4 - DB

Fig. 3. Effect of 2,4-DB on succinate oxidation. A: method I application. B: method II

application. Mitochondria - 0.6mg protein, succinate - 7 mM, ADP - 165 p.M.
 170 M. Budimir e t al.

and ADP (Fig. 4A)_ Fifty percent inhibition of state 3 and state 4 respiration was
induced by 3 • 10-4M and 1.6 • 10-3M, respectively. Similar results were obtained
in experiments with incubation of 2,4-DB with mitochondria (Fig. 4B).

Table I shows the effects of captan and 2,4-DB on respiratory control ratio and
ADP/O ratio. Respiratory control ratio and ADP/O ratio were decreased when
captan and 2,4-DB concentrations were increased.

Effects on NADHz: cytochrome c oxidoreductase. The effects of captan and

2,4-DB on NADH2 cytochrome c oxidoreductase were examined (using the inner
mitochondrial membrane preparation) in order to locate more closely the site of their
action. For comparison, rotenone, an inhibitor of NADH~: cytochrome c oxidoreduc-
tase was used. The results presented in Table II indicates that 50% inhibition of
enzymatic activity was obtained by 8.4 x 10-SM rotenone, 3.3 x 10-SM captan and
3.3 • 10-4M 2,4-DB.

Effects on photochemical reactions of chloroplasts. Effects of examined com-

pounds on several photoreactions of isolated chloroplasts are presented in Table III.
With water as the electron donor and ferricyanide as the electron acceptor the rate of
oxygen evolution was measured under both nonphosphorylating and phosphorylating
conditions. The rates of noncyclic photophosphorylation and ADP/O ratios were
also determined.

Malate oxidation

O2/min /dM O2/nnin

35 35

3O A 30.

25 25.

20 20:

15 15"

lO12
IV 1
0~.~ . . . . . . ~ 0
1.0 3.3 6.6 10 16 .0 3.3 6'.6 1'0 ;6
X 10--4M X 10--4M
2,4 - DB

Fig. 4. Effect of 2,4-DB on malate oxidation. A: method I application. B: method II

application. Mitochondria - 0.6 mg protein, malate - 30 mM, ADP - 165 /aM.
 Effects of 2,4-DB and Captan 171

T a b l e I. Effects of captan and 2,4-DB on oxidative phosphorylation in white potato

mitochondria
Concentra-
tion of
pesticides a Substrate ADP/O RC Substrate ADPJO RC
{ x 10-4/14) Succinate Malate

Captan

0 (DMSO) 1.40 2.22 3.38 2.50

0.33 1.39 1.28 0.00 1.00
0.66 1.26 1.92 0.00 1.00
1.00 1.03 1.70 0.00 1.00
1.60 1.34 1.80 0.00 1.00
2.00 1.21 1.40 0.00 1.00
2.60 0.00 1.00 0.00 1.00
2,4-DB

0 (methanol) 1.50 2.39 2.35 2.29

0.33 1.37 2.77 2.52 2.50
0.66 1.32 1.64 2.13 2.01
1.60 1.79 1.52 1.39 1.34
3.30 1.20 1.36 0.00 1.00
6.60 1.10 1.00 0.00 1.00
I0.00 0.90 1.00 0.00 1.00
16.00 0.00 1.00 0.00 1.00

a Pesticides were added to the reaction mixture in second state 4, after substrate and ADP

Captan inhibited basal electron transport (with ferricyanide) up to 30% at 0.5 x

10-3M. Both ADP- and a m m o n i u m c h l o r i d e - s t i m u l a t e d rates o f o x y g e n evolution
were inhibited to the same extent (35-40% inhibition by 0.5 x 10-3M captan).
A d d i t i o n o f captan stimulated o x y g e n evolution in the A D P - l i m i t e d state. The rate
o f A T P generation was inhibited b y increasing concentrations of captan (50%
inhibition at 0.25 x 10-3M), while the A D P / O ratio averaged 1.0.

T a b l e I I . Effects of captan and 2,4-DB on NADH2: cytochrome c

oxidoreductase activity

Pesticide I5oa

Rotenone 8.44 x 10-6M

Captan 0.33 x 10-4M
2,4-DB 3.30 x 10-4M
a I50 x Molar concentration required to inhibit the reaction by 50%
172 M. Budimir et al.

T a b l e III. Effects of captan and 2,4-DB on photochemical reactions of chloroplasts

Concentration Rate of O., evolution a

of pesticides ADP ADP NH4CI
Basal stimu- limi- stimu-
( x 10-4M) (FeCN) lated ted lated AATP ADP/O

Captan
0 (DMSO) 28.1 43.4 20.9 72.3 99 1.10
0.33 28. i 42.0 24.1 69.8 87 0.98
0.66 28. I 40.1 24.1 61.8 77 0.90
1.20 24.5 37.6 24.5 61.3 66 0.94
1.50 24.5 34.3 24.5 61.3 60 0.85
2.00 23.6 34.2 25. I 57.1 57 0.80
3.00 21.3 31.0 25.3 49.8 43 0.88
5.30 19.4 28.2 27.3 42.4 39 0.89
2,4-DB
0 (methanol) 31.6 49.7 18.9 78.9 126 1.20
0.33 28.6 42.6 17.3 65.5 106 1.20
0.66 26.8 41.3 20.2 66.2 115 1.20
1.30 25.3 41.8 18.1 71.1 106 1.20
6.60 25.4 36.9 17.3 67.1 92 1.20
16.60 19.7 30.0 118 55.2 63 1.I0
33.00 17.3 22.1 11.8 41.8 51 1.20

a/zmoles of product formed/mg chlorophyll/hr

2,4-DB also inhibited oxygen evolution with ferricyanide as the electron acceptor;
I • 10"3M herbicide produced 30% inhibition while 3 x 10-3M herbicide produced
40% inhibition of basal electron transport. The inhibition of ADP-stimulated, ADP-
limited and NH4C1 stimulated oxygen evolution increased with increasing concentra-
tion of herbicide; inhibition of approximately 50% was reached at a concentration of
3.3 x 10-3M. Noncyclic photophosphorylation was inhibited by 50% at 1.7 x
10-3/14 herbicide, while ADP/O ratio remained at 1.2.

Discussion

The results presented here show that '2,4-DB and captan can inhibit electron
transport and phosphorylation in isolated mitochondria and chloroplasts, which is in
agreement with results by some other authors (Nelson 1971, Matlib and Kirkwood
1972, Moreland and Hill 1962, Chkanikov et al. 1966). Captan and 2,4-DB inhibited
state 3 respiration of mitochondria with both malate and succinate as substrates.
 Effects of 2,4-DB and Captan 173

State 4 respiration was also inhibited when malate was used as substrate, indicating
that both 2,4-DB and captan acted as electron transport inhibitors.

With succinate as substrate, there was a stimulation of state 4 respiration by both

pesticides indicating their behavior as uncouplers. Similar to 2,4-dinitrophenol
(Ikuma and Bonnet 1967) maximal uncoupling was obtained when pesticide was
added after substrate and ADP; inhibition of oxygen uptake occurring at prolonged
time of pre- incubation and at higher pesticide concentration (method B). However,
the limited stimulation of state 4 respiration with succinate as substrate and rela-
tively slow decrease of ADP/O ratio implicates marginal direct interference with the
ATP generation pathway. Differences in the succinate oxidation pattern, when
pesticides were applied with and without previous incubation with mitochondria,
may be relatedto the location of the reactive sites and to the ability of the inhibitors
to penetrate and partition into the mitochondria.

Much higher sensitivity of malate oxidation suggests that this portion of the
respiratory chain was more readily accessible to tested compounds than components
associated with succinate oxidation. This is supported by the effect of captan and
2,4-DB on the activity of NADH2: cytochrome c oxidoreductase; the enzymatic
activity was inhibited by 50% by the same concentrations of pesticides that induced
50% inhibition of malate oxidation. This also means that the inhibition of malate
oxidation is mainly due to the inhibition of NADH2:cytochrome c oxidoreductase.

2,4-DB and captan interfered with noncyclic electron transport in chloroplasts.

They inhibited oxygen evolution with ferricyanide as the electron acceptor, in the
phosphorylating and uncoupled state. They also inhibited the rate of ATP generation
by noncyclic photophosphorylation, but they did not affect significantly the ADP/O
ratio. The compounds differed in the degree by which they affected the mentioned
processes and also in the way they influenced ADP-limited electron transport. While
captan stimulated ADP-limited oxygen evolution, 2,4-DB inhibited it. In general, of
the two, captan was a more efficient uncoupler as well as a more efficient inhibitor
of electron transfer processes in mitochondria and chloroplasts.

The results obtained suggest that if the compounds should partition into the
chloroplasts and mitochondria in vivo, oxidative and photosynthetic phosphorylation
would be inhibited. The degree of inhibition would depend on the compound and on
its concentration.

References

Bonner, W. D. Jr.: General method for the preparation of plants mitochondria. In

Estabrook, R. W., and W. E. Pullman (eds): Methods in enzymology. Vol. X,
p. 126. New York: Academic press (1967).
 174 M. Budimir et al.

Chance, B., and C. R. Williams: The respiratory chain and oxidative phosphoryla-
tion. In Nord, F. F. (ed.): Advances in enzymology, Vol. XVII, P. 65. New
York: Intersc. Publishers (1956).
Chkanikov, D. I., V. I. Cercuskii, and V. I. Kostina: Vliyanie galoidfenoksikislot
na ciklicheskoe fotosinteticheskoe fosforilirovanie. Khimia v sel skom hozy. 4,
50 (1966).
Delieu, T. and D. A. Walker: An improved cathode for the measurement of
photosynthetic oxygen evolution by isolated chloroplasts. New Phytol. 71,201
(1972).
Douce, R., C. A. Mannela, and W. D. Bonner: The external NADH dehydrogenases
of intact plant mitochondria. Biochem. and Biophys. Acta 292, 105, (1973).
Ikuma, H., and W. D. Bonner, lr.: Properties of higher plant mitochondria. II.
Effects of DNP, m-CI-CCP and oligomycin on respiration of mung bean
mitochondria. Plant Physiol. 42, 1400 (1967).
Matlib, M. A., and R. C. Kirkwood: Differential effect of selected phenoxy-acid
compounds on oxidative phosphorylation of mitochondria from Vicia faba L.
Journ. Exp. Bot. 23, 886 (1972).
Moreland, D. E., and K. L. Hill: Interference of herbicides with the Hill reaction of
isolated chloroplasts. Weeds 10, 229 (1962).
Nelson, B. D.: Action of the fungicides captan and folpet on rat liver mitochondria.
Biochem. Pharmacol. 20, 737 (1971).
Penner, D., and F. M. Ashton: Biochemical and metabolic changes in plants
induced by chlorophenoxy herbicides. Resididue Reviews 14, 39 (1966).
Siegal, M. R., and H. D. Sisler: Reactions of folpet with purified enzymes, nucleic
acid and subcellular components of Saccharomyces pastorianus. Phytopathol.
58, 1129 (1968).
Wain, R. L.: The behavior of pesticides in the plant in relation to selectivity. In L.
J. Audus (ed.): The physiology and biochemistry of herbicides, p. 465. Lon-
don and New York: Academic Press (1964).
Walker, D. A.: Photosynthetic activity of isolated pea chloroplasts. In T. W.
Goodwin (ed.): Biochemistry of chloroplasts, Vol. II, p. 53. London and New
York: Academic Press (1967).
Wedding, R. T., and M. K. Black: Response of oxidation and coupled phosphoryla-
tion in plant mitochondi'ia to 2,4-dichlorophenoxyacetic acid. Plant Physiol.
37, 364 (1962).
West, K. R., and J. T. Wiskich: Photosynthetic control by isolated pea chloroplasts.
Biochem. J. 109, 527 (1968).