Comparative effects of the Roundup and glyphosate on mitochondrial oxidative phosphorylation
Abstract
The potential toxicity of the herbicide Roundup and its fundamental substance (glyphosate) was tested in bioenergetic functions of isolated rat liver mitochondria. Roundup stimulates succinate-supported respiration twice, with simultaneous collapse of transmembrane electrical potential, while glyphosate used in the same concentrations does not induce any significant effect. Additionally, Roundup depresses state 3 respiration by about 40%, at 15 mM, whereas uncoupled respiration in the presence of FCCP is depressed by about 50%. Depression of uncoupled respiratory activity is mediated through partial inhibition of mitochondrial complexes II and III, but not of complex IV. The phosphorylative system was affected by both a direct and an indirect effect on the F0F1 ATPase activity. The addition of uncoupled concentrations of Roundup to Ca2+-loaded mitochondria treated with Ruthenium Red resulted in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Therefore, the uncoupling of oxidative phosphorylation is also related to the non-specific membrane permeabilization induced by Roundup. Glyphosate alone does not show any relevant effect on the mitochondrial bioenergetics, in opposition to Roundup formulation products. The differences in the toxicity observed could be either attributed to some products of Roundup or to a synergic effect of glyphosate and formulation products. Bearing in mind that mitochondria is provided with a variety of bioenergetic functions mandatory for the regulation of intracellular aerobic energy production and electrolyte homeostasis, these results question the safety of Roundup on animal health.
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Available from: Francisco Peixoto
Comparative effects of the Roundup and glyphosate
on mitochondrial oxidative phosphorylation
Francisco Peixoto
*
Departamento de Quı
´
mica, Universidade de Tra
´
s-os-Montes e Alto Douro, 5001 Vila Real, Portugal
Received 7 September 2004; received in revised form 24 February 2005; accepted 16 March 2005
Available online 26 April 2005
Abstract
The potential toxicity of the herbicide Roundup and its fundamental substance (glyphosate) was tested in bioener-
getic functions of isolated rat liver mitochondria. Roundup stimulates succinate-supported respiration twice, with
simultaneous collapse of transmembrane electrical potential, while glyphosate used in the same concentrations does
not induce any significant effect. Additionally, Roundup depresses state 3 respiration by about 40%, at 15 mM, whereas
uncoupled respiration in the presence of FCCP is depressed by about 50%. Depression of uncoupled respiratory activity
is mediated through partial inhibition of mitochondrial complexes II and III, but not of complex IV. The phosphory-
lative system was affected by both a direct and an indirect effect on the F
0
F
1
ATPase activity. The addition of uncoupled
concentrations of Roundup to Ca
2+
-loaded mitochondria treated with Ruthenium Red resulted in non-specific mem-
brane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Therefore, the uncou-
pling of oxidative phosphorylation is also related to the non-specific membrane permeabilization induced by
Roundup. Glyphosate alone does not show any relevant effect on the mitochondrial bioenergetics, in opposition to
Roundup formulation products. The differences in the toxicity observed could be either attributed to some products
of Roundup or to a synergic effect of glyphosate and formulation products. Bearing in mind that mitochondria is pro-
vided with a variety of bioenergetic functions mandatory for the regulation of intracellular aerobic energy production
and electrolyte homeostasis, these results question the safety of Roundup on animal health.
2005 Elsevier Ltd. All rights reserved.
Keywords: Roundup; Glyphosate; Mitochondrial bioenergetics; Oxidative phosphorilation; Mitochondrial permeability transition
1. Introduction
Glyphosate, N-(phosphonomethyl)glycine (Fig. 1),
the active ingredient of very well-known herbicide prep-
arations, such as Roundup, is a systemic and non-selec-
tive herbicide used to kill broadleaved grass, and sedge
species (Williams et al., 2000). Although glyphosate is
already one of the most used xenobiotics in modern agri-
culture, we should expect an increasing utilization of
glyphosate largely due to the number of transgenic
plants developed to be tolerant to this herbicide (May
et al., 2002; Nadler-Hassar et al., 2004; Stephenson
et al., 2004) and due to the removal of patent protection
for Roundup by Monsanto Co. GlyphosateÕsLD
50
in
rats is greater than 4320 mg/kg of the body weight
(EPA, 1993). The effects of glyphosate on hepatic and
intestinal drug metabolizing enzyme activities studied
in rats, intragastrically exposed for two weeks, revealed
0045-6535/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2005.03.044
*
Tel.: +351 259350273; fax: +351 259350480.
E-mail address: fpeixoto@utad.pt
Chemosphere 61 (2005) 1115–1122
www.elsevier.com/locate/chemosphere
that glyphosate decreased the hepatic level of cyto-
chrome P-450 and monooxygenase activities as well as
the intestinal activity of aryl hydrocarbon hydroxylase
(Hietanen et al., 1983).
Although many studies have shown that glyphosate
has a low toxicity (Marc et al., 2002; Howe et al.,
2004; Pieniazek et al., 2004), Barbosa et al. (2001) re-
ported that a man accidentally sprayed with glyphosate
developed a symmetrical Parkinson syndrome. Some
authors have reported an increased risk for non-Hodg-
kinÕs lymphoma (NHL) following exposure to certain
pesticides (e.g. Roundup) (Hardell et al., 2002; De Roos
et al., 2003). Virtually, every pesticide product contains
ingredients other than those identified as the ‘‘active’’
ingredient(s), i.e. the one designed to provide the killing
action. These ingredients are misleadingly called ‘‘inert’’.
Commercial glyphosate formulations are more acutely
toxic than glyphosate, since the amount of Roundup re-
quired to kill rats is about 1/3 of the amount of glyphos-
ate alone (Martinez and Brown, 1991). Similar results
have been obtained in cell division, thus indicating a
synergy between glyphosate and Roundup formulation
products (Marc et al., 2002).
Electron microscopy of fish (Cyprinus carpio) ex-
posed by emersion in Roundup revealed that the herbi-
cide caused the appearance of myelin-like structures in
carp hepatocytes, the swelling of mitochondria and the
disappearance of the internal membrane of mitochon-
dria (Szarek et al., 2000).
The present study constitutes the very first attempt to
describe the interaction of Roundup and glyphosate
with mitochondrial functions. These studies are relevant
to toxicology because most of the energy involved in cel-
lular metabolism and intermediary metabolic com-
pounds is generated at the expense of mitochondrial
respiration (Erecinska and Wilson, 1982). Interactions
of chemical compounds with mitochondrial functions
can, therefore, result in severe impairment of the general
metabolism.
2. Materials and methods
2.1. Isolation of rat liver mitochondria
Wistar rats (200–300 g) were starved overnight before
being killed by cervical displacement. The isolation was
performed by the conventional method (Gazzotti et al.,
1997), with minor modifications. The homogenization
medium contained 0.25 M sucrose, 5 mM Hepes (pH
7.4), 0.2 mM EGTA (ethyleneglycol-bis(b-aminoethyl
ether) N,N,N
0
,N
0
-tetraacetic acid) and 0.1% fatty acid-
free bovine serum albumin (BSA). EDTA (ethylene-
diaminetetraacetic) EGTA and BSA were omitted from
the final washing medium, which was adjusted to pH
7.2. The final concentration of the mitochondrial protein
was determined by the biuret method (Gornall et al.,
1949) using BSA as standard. The experiments reported
here were carried out in accordance with the National
Requirements for Vertebrate Animal Research and the
European Convention for the Protection of Animals
used for Experimental and other Scientific Purposes.
2.2. Mitochondrial respiratory activity
Oxygen consumption of isolated mitochondria was
measured polarographically using a Clark-type oxygen
electrode (Ernster and Kuylenstierna, 1970) connected
to a suitable recorder in a closed water-jacketed 1.0 ml
chamber with magnetic stirring, at 25 C. Respiration
rates were calculated assuming an oxygen concentration
of 450 nAt O/ml in the experimental medium at 25 C.
The standard respiratory medium consisted of 130 mM
sucrose, 50 mM KCl, 5 mM MgCl
2
, 5 mM KH
2
PO
4
,
and 5 mM Hepes, pH 7.2. Roundup or glyphosate was
added in aliquots (a few microlitres), from a concen-
trated aqueous solution (1 M, adjusted to pH 7.0), to
1 ml of the standard respiratory medium supplemented
with mitochondria (0.8 mg protein) and allowed to incu-
bate for 5 min before the addition of respiratory sub-
strate, i.e., before the beginning of the respiratory
activity. In all the experiments, Roundup (mM) concen-
trations are expressed in terms of the final glyphosate
concentration presented in each assay. State 3 was
elicited by adding adenosine 5
0
-diphosphate (ADP;
1 mM), and uncoupled respiration by adding 1 lM
FCCP. The respiratory control ratio (RCR) and ADP
to oxygen ratio (ADP/O) were calculated according to
the method previously described (Chance and Williams,
1956), considering that the saturation oxygen concentra-
tion was 232 nmolO
2
/ml in the reaction medium at
25 C.
2.3. Membrane potential (Dw) measurements
The reactions were conducted under a continuous
stream of O
2
to avoid anaerobisis. The transmembrane
potential (Dw) was estimated with a TPP
+
electrode
according to the equation of Kamo et al. (1979), without
correction for the ‘‘passive’’ binding contribution of
TPP
+
to the mitochondrial membranes (as the purpose
of the experiment was to show relative changes in the
potential rather than absolute values). A matrix volume
of 1.1 ll/mg mitochondrial protein was considered and
HO C C
O
NC P OH
OH
O
H
H
H
H
H
Fig. 1. Structure of glyphosate.
1116 F. Peixoto / Chemosphere 61 (2005) 1115–1122
valinomycin was used to calibrate the basal line. Reac-
tions were carried out at 25 C in 1 ml of the standard
respiratory medium (the same medium as described
for the oxygen consumption experiments) supplemented
with 3 lM TPP
+
and 0.8 mg mitochondrial protein.
Calibration runs in the presence of both xenobiotics
excluded any direct interference in the electrode signal.
2.4. Enzymatic activities
Succinate dehydrogenase activity was measured spec-
trophotometrically by the reduction of DCIP (2,6-
dichlorophenolindophenol) at 600 nm in the presence
of PMS (phenazine methasulphate) (Singer, 1974). The
reaction was performed in 1 ml of the standard reaction
medium supplemented with 5 mM succinate, 2 lM rote-
none, 0.1 lg antimycin A, 1 mM KCN, 0.025% Triton
X-100 at 25 C and 0.5 mg protein of disrupted mito-
chondria (two cycles of freezing and thawing).
Succinate cytochrome c reductase activity was mea-
sured spectrophotometrically (Tisdale, 1967)at25C
by following the reduction of oxidized cytochrome c
by the increase in absorbance at 550 nm. The reaction
was initiated by the addition of 5 mM succinate to
2 ml of the standard reaction medium supplemented
with 2 lM rotenone, 1 mM KCN, 54 lM of cytochrome
c and 1 mg protein of broken mitochondria.
Cytochrome c oxidase activity was measured polaro-
graphically (Brautigan et al., 1978)at25Cin1ml
of the standard reaction medium supplemented with
5 mM succinate, 2 lM rotenone, 10 lM cytochrome c
and 0.5 mg protein broken mitochondria. The reaction
was initiated by the addition of 5 mM ascorbate plus
0.25 mM TMPD.
ATP-synthase activity was determined by monitoring
the pH increase associated with ATP synthesis (Madeira
et al., 1974). The reaction was carried out in 2 ml of the
reaction medium containing 130 mM sucrose, 50 mM
KCl, 5 mM MgCl
2
and 2 mM KH
2
PO
4
(pH 7.2), supple-
mented with 5 mM succinate and 0.5 mg of protein. The
reaction was initiated by the addition of 200 lM ADP to
the mitochondrial suspension. The pH change was eval-
uated with a Crison pH meter connected to a Perkin–
Elmer recorder. The addition of oligomycin (2 lg/mg
protein) completely halted H
+
consumption. H
+
con-
sumption was calculated after an elapsed time of 2 min
from the start of the reaction.
ATPase activity was determined by monitoring the
pH change in association with ATP hydrolysis, as previ-
ously described (Madeira et al., 1974). The reaction was
carried out at 25 C, in a 2 ml reaction medium (130 mM
sucrose, 50 mM KCl, 5 mM MgCl
2
, 0.5 mM HEPES,
2 lM rotenone, pH 7.2) and freeze thawed mitochondria
(0.5 mg) were added. The reaction was initiated by the
addition of 2 mM Mg-ATP; production of H
+
was mea-
sured after an elapsed time of 3 min from the start of the
reactions, following calibrations of the trace with ali-
quots of titrated HCl. The addition of oligomycin
(1 lg/mg protein) to the medium completely halted the
production of protons.
2.5. Mitochondrial swelling
Mitochondrial osmotic swelling was monitored by
detecting turbidity, at 520 nm with a suitable spectro-
photometer-recorder set up. The reactions were carried
out at 25 C in 2.0 ml of the required media as indicated
in legends to figures.
2.6. Chemicals
All reagents were of analytical grade for research.
Glyphosate was supplied by the Monsanto Company
and Roundup was purchased in a specialized store.
2.7. Treatment of the data
Mean results are presented with the in respective
±SD values from at least three independent experiments.
Statistical analyses were performed using two-tailed
unpaired t-tests. A p value <0.05 was considered statis-
tically significant.
3. Results
3.1. Xenobiotic effects on the mitochondrial respiration
The effects of Roundup and glyphosate on succinate-
dependent respiratory indexes, RCR and ADP/O ratio
of rat liver mitochondria are shown in Table 1. Glyphos-
ate up to 5 mM does not significantly affect the RCR
and ADP/O. However, even at 0.5 mM (concentrations
are expressed in terms of the final glyphosate concentra-
tion present in each assay) Roundup significantly
depresses RCR and ADP/O ratio.
Fig. 2 shows the effects of Roundup and glyphosate
on the respiratory rates characteristic of state 4 (succi-
nate alone), ADP-stimulated respiration (state 3), and
FCCP-stimulated respiration (uncoupled respiration)
of rat mitochondria. Glyphosate did not show any sig-
nificant effect on the respiratory rates. State 3 and
uncoupled respiration rates were significantly depressed
by Roundup for concentrations up to 15 mM, whereas
state 4 respiration was stimulated twice as much.
Control experiments show that in the absence or in
the presence of any of the used xenobiotics oxygen con-
sumption was completely inhibited by the addition of
antimycin A (not shown), indicating that the oxygen
consumption occurred exclusively due to the respiratory
activity.
F. Peixoto / Chemosphere 61 (2005) 1115–1122 1117
3.2. Xenobiotic effects on mitochondrial membrane
potential
The effects of Roundup and glyphosate on the ener-
gization and phosphorylation capacities of mitochon-
dria were investigated through following the
transmembrane potential (Dw) developed by mitochon-
dria upon succinate oxidation (Fig. 3A). The presence
of glyphosate up to 15 mM did not affect the Dw;on
the other hand, up to 10 mM Roundup almost col-
lapsed Dw promoted by succinate although the dissipa-
tion, is already very significant for a concentration of
1 mM. After succinate addition, mitochondria devel-
oped a transmembrane potential of about 210 mV.
Addition of ADP to succinate energized mitochondrial
suspension causes a depolarization of Dw to about
190 mV, since ATP-synthase uses Dw to phosphory-
late ADP. After a short lag phase, during which ADP
Table 1
Effects of Roundup and glyphosate on succinate-dependent respiratory indexes RCR and ADP/O of rat liver mitochondria
(mM) Roundup Glyphosate
ADP/O RCR ADP/O RCR
0.00 1.81 ± 0.03 4.02 ± 0.04 1.82 ± 0.04 4.03 ± 0.05
0.50 1.56 ± 0.04
*
3.06 ± 0.08
*
1.81 ± 0.02 4.01 ± 0.06
1.00 1.53 ± 0.02
*
3.14 ± 0.06
*
1.80 ± 0.05 3.98 ± 0.09
2.00 1.48 ± 0.03
*
3.00 ± 0.06
*
1.81 ± 0.03 3.92 ± 0.10
5.00 1.13 ± 0.02
*
2.11 ± 0.09
*
1.80 ± 0.04 3.87 ± 0.08
The results correspond to the mean ± SD of four to six independent experiments.
*
p < 0.05 compared with the control (in the absence of xenobiotics).
0
50
100
150
200
250
0510
15 20
Oxygen consumption
(% control)
Xenobiotic (mM)
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Fig. 2. Effect of Roundup (fulfilled symbol) and glyphosate
(open symbol) on respiratory rates of mitochondria rat liver.
Mitochondria (0.8 mg) were incubated in 1 ml of the respiratory
standard medium, at 25 C, in the presence of used xenobiotic
concentration (0, 0.5, 1, 2, 5, 10 and 15 mM), for 5 min. State 4
respiration (j,h) was initiated by the addition of 10 mM
succinate. State 3 respiration (d,s) energized by 10 mM
succinate was initiated by the addition of 1.5 mM ADP, added
2 min after the initiation of state 4 respiration and FCCP-
uncoupled respiration (m,n). Values are the means ± SD of 4
independent experiments.
-200
-220
-180
-160
-140
-120
-100
-80
0 5 10 15 20
mitochondrial transmembrane potential (mV)
Xenobiotic (mM)
A
-210
-190
-181
-196
ADP
0
0.5
1
2
B
Fig. 3. Effect of Roundup (s) and glyphosate (h)on
membrane potential in rat liver mitochondria (A) and the effect
of Roundup on the phosphorylation rate of rat liver mitochon-
dria supported by succinate (B). Mitochondria (0.8 mg) were
incubated in 1 ml of the standard respiration medium supple-
mented with 3 lM TPP
+
and 2 lM rotenone for 5 min at 25 C
in the absence and presence of Roundup (0, 0.5, 1 and 2 mM),
before energization with 10 mM succinate. The phosporilation
was initiated with the addition of ADP (150 lM). Valinomycin
was added at the end of each assay to elicit complete collapse of
membrane potential.
1118 F. Peixoto / Chemosphere 61 (2005) 1115–1122
phosphorylation takes place, the transmembrane
potential repolarizes back to the initial value. The pres-
ence of Roundup in the reaction medium depresses Dw
promoted succinate. Furthermore, Roundup not only
decreases the depolarization and repolarization ampli-
tude induced by ADP, but also lengthens the lag phase
preceding repolarization (Fig. 3B), pointing to an inhi-
bitory effect on phosphorylation efficiency which is
also corroborated by the results obtained for ADP/O
(Table 1).
3.3. Effects of the xenobiotics on the mitochondrial
enzymatic activities
Studies of enzymatic activities of respiratory com-
plexes II, III and IV localized the components of the
mitochondrial respiratory chain affected by Roundup
and glyphosate. Glyphosate, in the concentration up to
15 mM, does not affect any of the enzymatic activities
of respiratory complexes (Fig. 4). Fig. 4 shows that the
terminal segment of the chain, cytochrome c oxidase
(complex IV), was not affected by Roundup; however,
succinate dehydrogenase (complex II) and succinate
cytochrome c reductase (complex III) were partially
inhibited, indicating that Roundup interacts with elec-
tron transfer at the level of complex II and III.
Fig. 5 shows the effects of glyphosate and Roundup
on the ATPase and ATP synthase activities. Glyphosate,
in the concentration up to 15 mM, did not show any sig-
nificant effect on the ATPase and ATPsynthase activi-
ties. For concentrations up to 15 mM Roundup
significantly depresses ATPase activity (60%), whe-
reas up to 5 mM ATP synthase was almost inhibited
(91%).
3.4. Mitochondrial swelling
In order to evidence the possible protonophoric
properties of the tested xenobiotics, mitochondrial swell-
ing experiments were performed in iso-osmolar medium
in the presence of Roundup or glyphosate. The K
+
ion-
ophore valinomycin was added in order to allow the
movement of K
+
across the mitochondrial membrane.
As illustrated in Fig. 6, under these experimental condi-
tions, both Roundup (A) and glyphosate (B) do not in-
duce a significant swelling. In fact, only for 15 mM of
Roundup was the permeability of mitochondria protons
slightly increased, a small effect compared with that of
FCCP.
Fig. 7 shows that unlike glyphosate, Roundup, in-
duces non-specific mitochondrial membrane perme-
abilization. The Ca
2+
concentration that should be
applied to assess the effect of Roundup on PTP induc-
tion was evaluated and a Ca
2+
concentration of 50 lM
was selected. In the presence of Roundup, Ca
2+
-loaded
mitochondria treated with RuRed underwent a per-
meability transition, as reflected by the decrease in
absorbency at 520 nm, an effect that was completely
inhibited by prior addition of Cyclosporine-A (CsA)
(Zoratti and Szabo
`
, 1995). Treatment of mitochondria
with glyphosate concentrations up to 15 mM did not
induce any swelling.
50
60
70
80
90
100
110
120
0 5 10 15 20
Enzymatic activities
(% of control)
Xenobiotic (mM)
*
*
Fig. 4. Effect of Roundup (fulfilled symbol) and glyphosate
(open symbol) on the respiratory complexes: succinate dehy-
drogenase (d,s), succinate cytochrome c reductase (j,h) and
cytochrome c oxidase (m,n). Mitochondria (0.8 mg) were
incubated in 1 ml reaction mediums (described in methods),
at 25 C. Assays were performed in the conditions described in
methods. Values are the means ± SD of 4 independent
experiments.
0
20
40
60
80
100
120
-5 0 5 10 15 20
ATPase and ATPsynthase
(% of control)
Xenobiotic (mM)
*
*
Fig. 5. Effect of Roundup (fulfilled symbol) and glyphosate
(open symbol) on ATP synthase (d,s) and ATPase (j,h). The
activities were determined as described in Materials and
Methods. Values are means ± SEM of three to five independent
experiments. *Values statistically different from control
(p < 0.05).
F. Peixoto / Chemosphere 61 (2005) 1115–1122 1119
4. Discussion
The present study addresses the in vitro effect of
Roundup and glyphosate on rat liver mitochondrial bio-
energetics. Liver mitochondria preparation is a very con-
venient method for studying bioenergetic toxicities of
xenobiotics (Da Silva et al., 1998; Peixoto et al., 2003);
moreover, data from mitochondrial studies can gener-
ally be correlated with cytotoxicity parameters evaluated
by other methods (Blondin et al., 1987; Knobeloch et al.,
1990a,b).
Data obtained in this study clearly demonstrate the
ability of Roundup to impair mitochondrial bioenergetic
reactions. Alteration of basic mitochondrial functions
was monitored by the detection of changes induced in
mitochondrial respiration and membrane energization
(Dw). Form the chemical structure of glyphosate, a pro-
tonophore action promoted by the phosphonic group
cannot be taken into consideration since pK
a1
(2.0)
and pK
a2
(2.25) values are too low (Wauchope, 1978).
Nevertheless, the carboxylic group, which has a pK
a
(5.5) (Wauchope, 1978) value near that of acetic acid
(acetic acid at pH 7.1 can transport protons across the
membrane), could not participate in the transmembrane
transport of protons, since at pH 7.1 the molecule of gly-
phosate should be almost charged ([H
3
A] ’ 7.9 · 10
6
[H
2
A
]). As a result, glyphosate is not capable of
crossing the lipidic membrane. Consequently, is not sur-
prising that under these experimental conditions gly-
phosate does not have any significant effect on the
mitochondrial state 4, on state 3, and on uncoupled res-
piration, whereas Roundup inhibited the state 3 and the
uncoupled respiration. State 4 stimulation by Roundup
was not higher since two antagonistic effects are ob-
served; a mitochondrial membrane permeability which
increases the respiratory rate and an inhibition of the
succinate oxidation, which decreases the respiratory
rate. From Fig. 2 (state 4 respiration in the presence of
Roundup) we can see that the inhibitory effect is prepon-
derant at higher concentrations.
A similar result was reported for potato mitochon-
dria, where a pre-incubation period in the presence of
glyphosate 0–50 mM did not reveal any measurable ef-
fect (Arnaud et al., 1993). However, with rat liver mito-
chondria glyphosate at a concentration of 50 mM
reveals a small but significant inhibition on the mito-
chondrial bioenergetics (data not shown).
Val
Glyphosate/ FCCP
0
10
15
FCCP
Val
Roundup/ FCCP
0
10
15
FCCP
= 0.05A
520 nm
20 sec
Fig. 6. Mitochondrial swelling induced by Roundup and glyphosate on valinomycin-treated mitochondria incubated in hyposmotic
K
+
-acetate medium. Rat liver mitochondria (0.3 mg/ml) were added to reaction medium containing 54 mM K
+
acetate, 5 mM HEPES-
Na
+
buffer pH 7.1, 0.1 mM EGTA, 0.2 mM EDTA, 15 lM atractyloside, 1 lM antimycin A, 100 lMNa
+
azide, 200 lM propranolol
and 0.1% BSA. Valinomycin (1 lM) was added where indicated. Roundup or glyphosate was added at the concentrations of 0
(control), 10 and 15 mM. The traces are representative of a group of at least three independent experiments.
RuRed
Roundup/glyphosate
= 0.1A
520 nm
2 min
b
c
d
a
,
, a, A, B, C, D
Fig. 7. Mitochondrial permeability transition induced by
Roundup or glyphosate on Ca
2+
-loaded (50 lM) mitochondria
treated with ruthenium red. Rat liver mitochondria (0.5 mg/ml)
were added to reaction medium (250 mM sucrose, 10 mM
HEPES-Na
+
buffer pH 7.2, 1 mM KH
2
PO
4
, supplemented with
4 lM rotenone, 0.5 lg oligomycin/ml and energized with 5 mM
succinate, at 25 C) in the presence of: (line a
0
) 1 M CsA, Ca
2+
and Roundup 15 mM. Ruthenium Red (1 lM) was added
where indicated, followed by an addition of Roundup or
glyphosate in the following concentrations: 0 mM, (line a, A)
5 mM, (line b, B) 10 mM, (line c, C) and 15 mM, (line d, D).
1120 F. Peixoto / Chemosphere 61 (2005) 1115–1122
The in vitro effect of glyphosate on rat liver mito-
chondria is not completely concurrent with the results
obtained in vivo (Olorunsogo et al., 1979). For in vitro
assays, higher concentrations or a longer incubation per-
iod should probably be used in order to obtain a better
correlation between in vivo and in vitro results. How-
ever, the assays with viable mitochondria cannot be
longer than a few hours, which means that only acute
effects can be studied.
Fig. 3A shows that Roundup promotes a strong de-
crease in mitochondrial transmembrane (Dw) potential
in a dose-dependent manner, with a maximal effect
for 10 mM. Meanwhile glyphosate, even at 15 mM,
does not affect the Dw, which is in agreement with
the results obtained for oxygen consumption. The
observed decrease in the Dw induced by Roundup can-
not be attributed to a protonophoric action, since
the valinomycin-induced swelling in KCl medium was
not significantly induced by Roundup or glyphosate.
Under these experimental conditions, the obtained re-
sults are not concurrent with the reported for Cyprinus
carpio which were exposed by emersion in Roundup
(Szarek et al., 2000). This should not come as a sur-
prise though reported comparisons between species
showed that the toxicity of Roundup or glyphosate
varied according to species and developmental stage
(Howe et al., 2004). The Dw collapse promoted by
Roundup could not be explained by a protonophoric
action but it was certainly the result of a non-specific
membrane permeabilization, referred to as mitochon-
drial permeability transition (MPT) (Bernardi, 1992)
specifically inhibited by CsA (Zoratti and Szabo
`
,
1995) and due to the inhibition of the complex II
and III.
Roundup depresses the efficiency of the electron
transport chain when succinate is the oxidable substrate,
but does not affect the terminal segment of the chain.
However, succinate dehydrogenase and succinate cyto-
chrome c reductase are significantly inhibited by Round-
up, suggesting that this herbicide affects the redox
electron transfer chain at the level of complexes II and
III (Fig. 4), although an effect on the translocation of
succinate across the inner membrane cannot be over-
looked. Glyphosate does not affect any of the studied
respiratory complexes (Fig. 4).
The results obtained for ATP synthase (ATPase) sys-
tem (Fig. 5) indicate that Roundup, though not glyphos-
ate, at concentrations up to 15 mM, can directly interact
with the enzymatic complex (ATPase/ATP synthase).
However, from the difference observed between the inhi-
bition promoted on the ATPase activity and in ATP syn-
thase, it can be concluded that a significant part of the
inhibition observed on the ATP synthase is merely the
result of the inhibitory effect on the respiratory com-
plexes II and III and due to non-specific inner mitochon-
drial membrane permeabilization.
Glyphosate presents a low partition coefficient in the
mitochondrial membrane; considering that the greater
part of the phosphorilative reactions are performed in
the membrane. Any effect is the result of a much less
xenobiotic concentration than that used in the assay.
In fact, in potato tuber mitochondria incubated for
15 min in a medium containing 50 mM glyphosate, the
membrane concentration may reach a maximum of
5 lM(Arnaud et al., 1993). It is, therefore, easy to
understand why biochemical processes taking place
inside the membrane (vectorial transport of protons,
electron transfer) are not significantly affected by
glyphosate.
The observed alterations in mitochondrial bioener-
getics caused by Roundup cannot be exclusively attrib-
uted to the active ingredient, but may as well be the
result of other chemicals e.g. POEA, or due the possible
synergy between glyphosate and Roundup formulation
products. This synergy between glyphosate and Round-
up inert ingredients has also been reported in cell divi-
sion (Marc et al., 2002), where the delay observed in
the cell cycle could be the result of alterations on the
mitochondrial bioenergetic reactivityÕs, which haves
drastic consequences on cellular function through the
perturbation of the bioenergetic charge and balance of
the cell. Therefore, the reduced energetic efficiency of
mitochondria may account for some toxic effects result-
ing from the impairment of the energy requirements of
the cell and from the crucial importance of energy
metabolism in active tissues, e.g. liver.
In short, in this study, we found that the so called
‘‘inert ingredients’’ used in Roundup are greatly respon-
sible for the observed toxicity at the bioenergetic level.
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- "Although animals do not possess the physiological targets for specific glyphosate toxicity, it has been demonstrated that both the active ingredient and its commercial formulations affect physiological and biochemical parameters in animals. These effects include: an inhibition ⁎ ⁎ Corresponding author. of cell respiration (Peixoto, 2005), a reduction in acetylcholinesterase activity in mollusks and fish (Sandrini et al., 2013) and reproduction impairment in fish (Lopes et al., 2014). Besides these effects, it has been proposed that glyphosate exposure may cause alterations in enzymes of the antioxidant defense system in invertebrates such as annelids (Contardo-Jara et al., 2009) and insects (Aguiar et al., 2016). "
[Show abstract] [Hide abstract] ABSTRACT: Glyphosate based herbicides, including Roundup, are widely employed in agriculture and urban spaces. The objective of this study was to evaluate the toxicological effects of Roundup on the estuarine polychaeta Laeonereis acuta. Biomarkers of oxidative stress as well as acetylcholinesterase and propionilcholinesterase activities were analyzed. Firstly, the LC50 96 h for L. acuta was established (8.19 mg/L). After, the animals were exposed to two Roundup concentrations: 3.25 mg/L (non-observed effect concentration – NOEC) and 5.35 mg/L (LC10) for 24 h and 96 h. Oxygen consumption was determined and the animals were divided into three body regions (anterior, middle and posterior) for biochemical analysis. An inhibition of both cholinesterase isoforms were observed in animals exposed to both Roundup concentrations after 96 h. A significant reactive oxygen species (ROS) reduction was observed in the posterior region of animals in both periods, while antioxidant capacity against peroxyl radicals (ACAP) was reduced in the posterior region of animals exposed for 24 h. Considering the antioxidant defense system, both GSH levels and enzyme activities (catalase, superoxide dismutase, glutathione s-transferase, glutathione peroxidase and glutamate cysteine ligase) were not altered after exposure. Lipid peroxidation was reduced in all analyzed body regions in both Roundup concentrations after 24 h. Animals exposed to the highest concentration presented a reduction in lipid peroxidation in the anterior region after 96 h, while animals exposed to the lowest concentration presented a reduction in the middle region. Overall results indicate that Roundup exposure presents toxicity to L. acuta, causing a disruption in ROS and ACAP levels as well as affects the cholinergic system of this invertebrate species.- "Ранее показано сходное повреждение ультраструктуры митохондрий в гепатоцитах карпа Cyprinus carpio L. после кратковременной экспозиции (0,5–1 час) в глифосате концентрацией 200–400 мг/мм 3 [Szarek et al., 2000] и в сперматоцитах гуппи (глазчатой пецилии) Poecilia vivipara Bloch & Schneider, 1801 при действии Раундапа в концентрации 130 и 700 мкг/л при 96-часовой экспозиции [Harayashiki et al., 2013]. Выявленные изменения свидетельствуют о нарушении процессов окислительного фосфорилирования, что подтверждается ингибированием in vitro и in vivo дыхательных ферментов и оксилительного потенциала мембран клеток более, чем на 40% в митохондриях гепатоцитов крыс [Peixoto, 2005] . Одновременное с повреждением митохондрий уменьшение запасов гликогена в цитоплазме гепатоцитов [Szarec et al., 2000] вызывает переход на гликолитический путь метаболизма , который предполагает снижение синтеза АТФ или повышенные энергозатраты на её производство, а также клеточную гипоксию. "
[Show abstract] [Hide abstract] ABSTRACT: Roundup (Roundup) is one of the most widely used in the world systemic non-selective herbicide, including the Russian. The active ingredient in the pesticide is glyphosate (N- (phosphonomethyl) glycine, C 3H8NO5P), which has long been considered low-toxic herbicide for animals. In 2015, glyphosate was attributed to the potentially dangerous carcinogens. It was found, the Roundup causes the mitochondria damage in gills, liver and kidney cells, the tissue oxidative stress, the kidney hyaline degeneration, the damage of the cell genetic apparatus, including germ cells, the increase in the hematocrit level, the number of red and white blood cells in fish. By electron microscopy the effect of sublethal concentration of Roundup (2 mg/l) was investigated for the first time on the leukocytes ultrastructure of immunocompetentce organs (kidney, spleen and liver) of young Amur sleeper — the invasive species in the ponds of the Upper Volga basin. It has been shown that the Roundup act in sublethal concentration (2 mg/l) at chronic 30-day effect as higher concentrations at acute action and lead to the damage of mitochondrial structure and the accumulation of glycogen granules in the granulocytes cytoplasm. The change in the number and size of specific granules in granulocytes (neutrophils and eosinophils) and occurred simultaneously with the appearance of phagosomes in the cytoplasm of cells were first revealed. It indicates an increase in nonspecific immunity reactions. The expansion of the rough endoplasmic reticulum channels in the plasma cells of the spleen and kidney indicates an increase of synthetic activity of plasma cells.- "Hence, our finding suggests that even low concentrations affected the growth of toad eggs and tadpoles and that the higher concentrated active ingredient might had already disappeared when the growth measurements were conducted. Again, not-declared adjuvants of glyphosatebased herbicide formulations with a higher toxicity than the glyphosate active ingredient itself (Perkins et al., 2000; Edginton et al., 2004; Howe et al., 2004; Peixoto, 2005; Wagner et al., 2013) or their properties as as endocrine disruptors (Gasnier et al., 2009; Defarge et al., 2016) might have contributed to the observed effects. Observed growth stimulations of toad larvae at low herbicide concentrations could perhaps be due to endocrine disruptors as they generally lack a dose-effect relationship (Vandenberg et al., 2012; Vandenberg, 2014). "
[Show abstract] [Hide abstract] ABSTRACT: Glyphosate-based herbicide formulations are broadly used in agriculture, silviculture, horticulture as well as in private gardens all over the world, thus posing the risk of potential contamination of nearby aquatic bodies inhabited by amphibians. Concurrently, climate change can be expected to alter the temperature of amphibian breeding sites. However, while either glyphosate-based herbicides or temperature have been shown to separately affect the development of amphibians, very little is known on possible interactive effects. We studied the impact of herbicide concentrations and temperature on growth and development of eggs and tadpoles of the Common toad (Bufo bufo L.). We hypothesized that (i) eggs would be better protected against herbicides than tadpoles because of their jelly coating, (ii) that higher temperatures would reduce potential herbicide effects because of an accelerated growth and a lower sensitivity of larger specimens. We conducted one experiment starting with eggs (Gosner stage, GS 9) and another experiment starting with tadpoles (GS 21-24) using a full factorial design with 5 concentrations of the herbicide formulation Roundup® LB Plus (0 mg acid equivalent L-1, 0.5 mg a.e. L-1, 1.0 mg a.e. L-1 or 1.5 mg a.e. L-1 and a pulse treatment with 3-4 times addition of 0.5 a.e. mg L-1 over the course of several weeks) and two temperature levels (15°C and 20°C). Contrary to our expectation, our results showed that toad eggs are more sensitive to herbicides than tadpoles leading to an averaged 31% increase in total length, tail length and body length compared to the herbicide-free control. Tadpole morphology, development or mortality was not influenced by herbicides. Higher temperature accelerated growth of both eggs and tadpoles. This is among the first study showing interactive effects between herbicides and temperature especially for egg development resulting in more pronounced herbicide effects at lower temperatures than at higher temperatures.
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