Microsoft word - glyphosate toxicity article.docx
Glyphosate Formulations Induce Apoptosis and Necrosis in Human Umbilical, Embryonic, and Placental Cells
Nora Benachour and Gilles-Eric Séralini*
University of Caen, Laboratory Estrogens and Reproduction, UPRES EA 2608, Institute of Biology, Caen 14032, France
Publication Date (Web): December 23, 2008
Copyright 2008 American Chemical Society
* To whom correspondence should be addressed. Tel: 33(0)2-31-56-56-84. Fax: 33(0)2-31-56-53-20. E-mail: email@example.com.
We have evaluated the toxicity of four glyphosate (G)-based herbicides in Roundup (R) formulations, from 105 times dilutions, on three different human
cell types. This dilution level is far below agricultural recommendations and corresponds to low levels of residues in food or feed. The formulations have
been compared to G alone and with its main metabolite AMPA or with one known adjuvant of R formulations, POEA. HUVEC primary neonate umbilical
cord vein cells have been tested with 293 embryonic kidney and JEG3 placental cell lines. All R formulations cause total cell death within 24 h, through an
inhibition of the mitochondrial succinate dehydrogenase activity, and necrosis, by release of cytosolic adenylate kinase measuring membrane damage. They also induce apoptosis via activation of enzymatic caspases 3/7 activity. This is
confirmed by characteristic DNA fragmentation, nuclear shrinkage (pyknosis), and nuclear fragmentation (karyorrhexis), which is demonstrated by DAPI in
apoptotic round cells. G provokes only apoptosis, and HUVEC are 100 times more sensitive overall at this level. The deleterious effects are not proportional
to G concentrations but rather depend on the nature of the adjuvants. AMPA and POEA separately and synergistically damage cell membranes like R but at
different concentrations. Their mixtures are generally even more harmful with G. In conclusion, the R adjuvants like POEA change human cell permeability
and amplify toxicity induced already by G, through apoptosis and necrosis. The real threshold of G toxicity must take into account the presence of adjuvants
but also G metabolism and time-amplified effects or bioaccumulation. This should be discussed when analyzing the in vivo toxic actions of R. This work
clearly confirms that the adjuvants in Roundup formulations are not inert. Moreover, the proprietary mixtures available on the market could cause cell damage and even death around residual levels to be expected, especially in
food and feed derived from R formulation-treated crops.
Humans are exposed daily to a great number of xenobiotics and their
metabolites, present as pollutants (1). They act as mixtures having compensatory, multiplicative, or synergistic effects, as we have shown (2) with
others (3, 4). The main glyphosate (G) formulations, commercialized as Roundup (R) from the Monsanto Company, are themselves already mixtures of
G and various adjuvants at different concentrations. We have studied these products, which are the major nonselective herbicides worldwide (5);
moreover, their use and presence in the food chain (6) are increasing since more than 75% of genetically modified edible plants have been designed to
tolerate high levels of these compounds (7). G and its major metabolite aminomethylphosphonic acid (AMPA) were classified among the first contaminants in rivers (8). The adjuvants, less measured in the environment,
are usually considered as inert and are protected as a “trade secret” in manufacturing (9). However, among them, the predominant one appears to be
the polyethoxylated tallowamine or POEA (10, 11), which has itself some toxicity (12), such as causing ocular burns, redness, swellings and blisters,
short-term nausea, and diarrhea. In combination with G, the mixture becomes more active (13). These products, like detergents, could allow facilitated G
penetration through plasmatic membranes, potentialization of its action, increased stability, and bioaccumulation (14, 15).
The dose- and time-dependent cytotoxicity of R Bioforce (360 g/L of G, R360)
on human placental and embryonic cells (15) could explain at least in part some reproductive problems (16). Among the two lines, 293 embryonic cells
have proven to be very suitable for estimating the hormonal activity for xenobiotics (17), and JEG3 cells are also considered a useful model for
examining placental toxicity (18). These lines may be equally or even less sensitive to xenobiotics than primary cultures (19). In the present study, we
also tested the mechanism by which R mixtures affect human primary cells of the umbilical vein cord endothelial cells (HUVEC) for comparative purposes.
The endothelial lining of blood vessels constitutes a permeable barrier between
the blood and the underlying tissues. The endothelium also plays an important role in various physiological processes, such as metabolism of vasoactive
substances and maintenance of antithrombotic factors on the vessel wall (20). The endothelial cells are exposed directly to chemicals circulating in the blood
of the umbilical cord and pass through the placenta (21). It is known that HUVEC cells may be a target for adverse effects of xenobiotics activated into reactive metabolites (22, 23). Other somatic cell types have been used to
study pesticide toxicity and apoptosis such as HeLa (24) and Jurkat (25), but none was before treated by glyphosate.
In human cells, we have demonstrated that G mixed with adjuvants in R360 was cytotoxic through alteration of succinate dehydrogenase SD (14, 15). With isolated rat liver mitochondria, it is demonstrated that R depresses the
mitochondrial complexes II (SD) and III (26). In sea urchin eggs, R deteriorated cell cycle checkpoints, and G with its adjuvants inhibited hatching
enzyme transcription synergistically (27, 28). Recently, it was shown in this model to activate the DNA damage checkpoint CDK1/cycline B of the first cell
cycle of development (29, 30) for commitment to cell death by apoptosis in the case of failure of DNA repair.
This work focuses on the cell death mechanism in human cells induced by four
different G formulations with a large number of agricultural applications. We have chosen Roundup Express (R7.2), Roundup Bioforce or Extra 360 (R360),
Roundup Grand Travaux (R400), and Roundup Grand Travaux Plus (R450) at subagricultural dilutions. We tested them on three important enzymatic
biomarkers. First, at the membrane level, we measured adenylate kinase (AK) activity after its release in the medium (31), revealing cytoplasmic membrane
rupture, corresponding to a necrosis and/or a secondary necrosis at the end of apoptosis (32). Second, at the mitochondrial respiration level, we measured
succinate dehydrogenase (SD) activity (33). Third, we tested the cytosolic level with caspase 3 and 7 activities to determine the apoptosis pathway (34-
36) and in situ DNA fragmentation (DAPI). Necrosis is evinced by cytoplasmic swelling, rupture of the plasma membrane, swelling of cytoplasmic organelles (particularly mitochondria), and some condensation of nuclear chromatin,
whereas apoptosis is manifested by cytoplasmic and nuclear condensation (pyknosis), nuclear fragmentation (karyorrhexis), normal morphological
appearance of cytoplasmic organelles, and an intact plasma membrane; following nuclear fragmentation, the cell disaggregates into a number of
membrane-bound apoptotic bodies (37, 32). By contrast, cell death is now known to be perpetrated through a variety of mechanisms. It can be classified
into four different types, based upon morphological characteristics: apoptosis (type 1), autophagy (type 2), necrosis (oncosis, type 3), and mitotic
The three human cell types allowed us to establish not only the differential sensitivity of these models but also the general human cell pathways of G-
based pesticides actions from 1 ppm (0.0001%); these were produced by G itself, its major metabolite AMPA, and the main adjuvant POEA, singly or in
N-Phosphonomethyl glycine (glyphosate, G, PM 169.07) and its major
metabolite AMPA (PM 111.04) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Herbicide Roundup formulations (Monsanto, Anvers,
Belgium) were available on the market: Roundup Express 7.2 g/L of G, homologation 2010321 (R7.2); Bioforce or Extra 360 at 360 g/L of G,
homologation 9800036 (R360); Grands Travaux 400 g/L of G, homologation 8800425 (R400); and Grands Travaux plus 450 g/L of G, homologation 2020448 (R450). A 2% solution of Roundup (1 or 2% is recommended by the
company for agricultural use) and an equivalent solution of glyphosate to Roundup Bioforce were prepared in serum-free medium and adjusted to pH 5.8
of the 2% Roundup Bioforce solution. The major adjuvant of Roundup, polyethoxylated tallowamine (POEA at 785 g/L), was a gift from Pr. Robert
Bellé (UMR 7150 CNRS/UPMC, Station Biologique de Roscoff, France). Successive dilutions were then obtained with serum-free medium. 4′,6′-
Diamidino-2-phenylindole, dihydrochloride (DAPI) nucleic acid stain powder was obtained from Lonza (Saint Beauzire, France). 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) and all other compounds, otherwise precised, were obtained from Sigma-Aldrich. MTT was prepared as a
5 mg/mL stock solution in phosphate-buffered saline, filtered through a 0.22 µm filter before use, and diluted to 1 mg/mL in a serum-free medium.
The human primary cells used in this work were HUVEC (C2519A) provided by
Lonza. Cells (passage 5 or 6) were grown according to the supplier, in specific endothelial growth medium EGM-2 SingleQuots (CC-4176) containing hEGF,
hydrocortisone, GA-1000 (Gentamicin, Amphotericin-B), FBS (fetal bovine serum), VEGF, hFGF-B, R3-IGF-1, ascorbic acid, and heparin. Fifty thousand
cells per well were grown at 37 °C (5% CO2, 95% air) over a 24 h period to 80% confluence in 48 well plates and were washed with serum-free EGM-2.
The human embryonic kidney 293 cell line (ECACC 85120602) and the human choriocarcinoma-derived placental JEG3 cell line (ECACC 92120308) were provided by CERDIC (Sophia-Antipolis, France). Cells were grown in phenol
red-free Eagle’s modified minimum essential medium (EMEM; Abcys, Paris, France) containing 2 mM glutamine, 1% nonessential amino acid, 100 U/mL
antibiotics (a mix of penicillin, streptomycin, and fungizone; Lonza), 10 mg/mL of liquid kanamycin (Dominique Dutscher, Brumath, France), and 10% FBS
(PAA, les Mureaux, France). The JEG3 cell line was supplemented with 1 mM sodium pyruvate. Fifty thousand cells per well were grown at 37 °C (5% CO2,
95% air) over a 48 h period to 80% confluence in 48 well plates and were washed with serum-free EMEM.
Cells were exposed for 24 h in serum-free medium to various dilutions of the
different treatments including the four Roundup formulations (R7.2, R360, R400, and R450), G, AMPA, or POEA (14 concentrations from 10 ppm to 2%)
and, particularly for POEA, were tested at the very low concentrations of 1 and 5 ppm; for AMPA, we tested in addition 4, 6, 8, and 10%. In another case,
cells were incubated with G, AMPA, and POEA mixtures by pairs at the final nontoxic dilution on SD of 0.5% on the human cell lines (293 or JEG3) and
0.05% on the human primary cells (HUVEC) in comparison to R360.
For the details, in each cell type, three combinations were studied. For the two cell lines, the first mixture was the combination of G (0.4999%) with POEA (0.0001%); the second was the combination of G (0.4%) with AMPA (0.1%),
and the third was AMPA (0.4999%) plus POEA (0.0001%). For the primary HUVEC cells, the first mixture was G (0.04999%) with POEA (0.0001%); the
second was G (0.04%) with AMPA (0.01%), and the third was AMPA (0.04999%) plus POEA (0.0001%).
This measure was based on the cleavage of MTT into a blue-colored product (formazan) by the mitochondrial enzyme succinate dehydrogenase (38, 39,
33); it was used to evaluate human cell viability. After cell treatments, the supernatants were recovered for the ToxiLight bioassay, and adherent cells
were washed with serum-free medium and incubated with 200 µL MTT per well after each treatment. The 48 well plates were incubated for 3 h at 37 °C, and
200 µL of 0.04 N hydrochloric acid-containing isopropanol solution was added to each well. The plates were then vigorously shaken to solubilize the blue formazan crystals formed. The optical density was measured at 570 nm using
a luminometer Mithras LB 940 (Berthold, Thoiry, France).
The bioluminescent ToxiLight bioassay (Lonza) was a nondestructive
cytotoxicity highly sensitive assay designed to measure toxicity in mammalian cells and cell lines in culture. It quantitatively measured the release of cytosolic
AK from the membranes of damaged cells (40, 31). AK is a robust protein present in all eukaryotic cells, which is released into the culture medium when
cells die, described as an important necrosis marker. The enzyme actively phosphorylated ADP, and the resultant ATP was then measured using the bioluminescent firefly luciferase reaction with the ToxiLight reagent. After 24 h
of different treatments, 50 µL of cell supernatants was deposited in 96 well black plates. Then, 50 µL of the AK detection reagent (AKDR) was added by
well. Plates were then placed under agitation for 15 min safe from the light, and then, luminescence was measured using the luminometer Mithras LB 940
(Berthold) at 565 nm. The serum-free medium was the negative control, and a positive control was the active reagent AKDR mixed with cells treated in the
serum-free medium to determine the basal activity.
The Caspase-Glo 3/7 assay (Promega, Paris, France) was a luminescent kit designed for automated high-throughput screening of caspases activity or
apoptosis. It can measure caspase 3 and 7 activities in purified enzyme preparations or cultures of adherent or suspension cells (41, 42, 36). The
assay provided a pro-luminescent caspase 3/7 substrate, which contains the tetrapeptide sequence DEVD active group. This substrate was cleaved to
release amino-luciferin, a substrate of luciferase used in the production of light. The Caspase-Glo 3/7 reagent was optimized for caspase activity,
luciferase activity, and cell lysis. The addition of the single Caspase-Glo 3/7
reagent, in an “add-mix-measure” format, resulted in cell lysis followed by caspase cleavage of the substrate and generation of a “glow type” luminescent signal. The Caspase-Glo 3/7 bioassay was carried out in 96 well white plates.
After cell cultures and their treatments by 50 µL of various dilutions, an equal volume of the reagent was added to each well. Plates were then agitated for
15 min safe from the light, to stabilize the light signal before measuring luminescence. Again, the negative control was the serum-free medium, and the positive control was the active reagent mixed with cells treated in the
serum-free medium to determine the basal activity of the caspases 3/7. Luminescence was measured using the luminometer Mithras LB 940 (Berthold)
At the end of the 24 h cell treatment, the serum-free medium was removed,
and cells were fixed in absolute ethanol−chloroform−acetic acid (6:3:1, v/v/v) for 1 day at −20 °C. Each well was washed with PBS (pH 7.4) and incubated
with 1 µg/mL DAPI solution (43). Staining of DNA with DAPI was examined with a microscope using a fluorescent mode (model Leïca LMD 6000, Rueil Malmaison, France). Labeled DNA of viable cells was scattered throughout the
nucleus, and bright condensation of chromatin revealed apoptotic cells (magnification, 400×). At the end of the cell treatment, the microphotographs
(magnification, 100×; blue filter) of cells without coloration were also obtained with the Leïca Microscopy Systems (model Leïca DC 100, Germany).
The experiments were repeated at least three times during different weeks on three independent cultures each time. All data were presented as the means ±
standard errors (SEs). Statistical differences were determined by a Student’s t test using significant levels of 0.01 (**).
We have studied for the first time the mechanism of cellular action of different
R on human cells, from placenta, embryonic kidney, and neonate. The first surprising results show that the four R herbicides and G cause cellular death
for all types of human cells, with comparable toxicity for each one but at different concentrations. For instance, 20 ppm for R400 at 24 h, the most
toxic, corresponds approximately to 47 µM G (8 ppm) with adjuvants (Figure 1). However, 4−10 ppm G alone is nontoxic; its toxicity begins around 1%.
The mechanism is constant for all R: There is a release of AK, indicative of cell membrane damage, and an inhibition of the mitochondrial SD (Figure 1). For
all R, the membrane damage (AK) is 1.5−2 times more sensitive than mitochondrial activity (SD) for 293 and JEG3 or equally sensitive for HUVEC.
By contrast, G induces mitochondrial toxicity without cell membrane damage. Unexpectedly, R400 is more toxic than another formulation containing more G, such as R450; the latter is in turn more harmful than R360, R7.2, and G in
last, but all of them are detrimental nonproportionally to the G concentration that they contain. This is illustrated in Figure 2.
Figure 1. Cytotoxic effects of four Roundup formulations (R) on three human
cell types. The R (from 10 to 2 × 104 ppm) contain different glyphosate (G) concentrations (7.2, 360, 400, or 450 g/L) and adjuvants. G alone was used as
control at equivalent quantities to R360 and at similar pH 5.8. The cells were either primary from neonate umbilical cord (HUVEC) or lines from embryo
(293) or placenta (JEG3). The actions on the mitochondrial succinate dehydrogenase (SD) activity (cellular viability in %, A) and on the release of
cytoplasmic adenylate kinase (AK) activity [cell death in relative luminescence units (RLU), B] were compared in serum-free medium after 24 h of exposure.
The 50% lethal dose (LD50) is indicated by a dashed line. SEs are shown in all instances (n = 12).
Figure 2. Nonlinear dose effects of R formulations. The LD50 (%) measured by
SD are compared for the 4 R (see the Figure 1A legend) and G for the three cell types in similar conditions.
The mitochondrial SD inhibition measures cell asphyxia. It is obvious from Figure 2 that 7.2 or 360 g/L G with adjuvants in R formulations has closely comparable actions on cell death, while 400 or 450 g/L gives inversely
proportional effects in another range. This is not an artifact since the embryonic and placental cell lines behave remarkably similarly in that regard
and the primary umbilical cord cells have sensitivity for all R and G just analogous to these cell lines (Figure 2). The mortality in all cases is not linearly
linked to G. The hypothesis that other substances are implicated has thus to be investigated in the formulation of the product.
Consequently, the major G metabolite, AMPA, and the surfactant POEA, the
main claimed adjuvant by the manufacturer (the exact composition is a secret of formulation), have been tested separately in a first approach, in comparison
to G and R360 as controls, and in similar conditions as in Figure 1, from very low subagricultural dilutions (10−6 if used pure like claimed by some farmers
and 10−4 if diluted as recommended at 1%).
G is claimed by the manufacturer to be the active ingredient, and it is claimed to be not toxic for human cells but toxic for vegetable ones when mixed with
inert components. Our study demonstrates for the first time that all products including AMPA and POEA provoke SD and AK effects in human cells, and thus
mortality (Figure 3), but at different concentrations. Astonishingly, the supposed inert product POEA is the most potent one. From 1 ppm, it begins to
alter SD in HUVEC and AK in 293 and JEG3. The mixture R is then more poisonous than G or AMPA. The metabolite AMPA itself destroys the cell
membrane (AK release), whatever the cell type. This is not observed with G, which is, however, 3−8 times more inhibitory on SD than AMPA, with some differences between cells. However, because the cell membrane damage is
generally more sensitive, the metabolite AMPA is finally more toxic than G on human cells. POEA is the most toxic; if it was the only adjuvant of R360, its
maximal concentration would be around 1−24 ‰, according to the cells. Thus, POEA could be considered as the active ingredient on human cell death and
more damaging than G. As R is more viscous than 1‰ POEA plus G, it is obvious that other compounds are in the mixture.
Figure 3. Cytotoxicity of R adjuvant (POEA) and glyphosate (G) metabolite (AMPA) on three human cell types. G and R360 were used as controls in
similar conditions as in Figure 1 (see legend), in comparison to R adjuvant POEA and G metabolite AMPA (1−105 ppm). The 50% lethal dose (LD50) is
indicated by a dashed line. SEs are shown in all instances (n = 12).
Thus, it was necessary to study the combined effects on cell membrane integrity (by AK release). We have tested the compounds by pairs at maximal
levels where alone they do not influence SD (Figure 4). This was to assess the respective role of each one, knowing that R contains all tested compounds
when metabolized. In contrast to previous results, the cells reacted differently. The mixtures were more disrupting on embryonic and umbilical cells, respectively, while placental carcinoma cells appeared to be more membrane-
resistant but to mixtures only. It is very clear that if G, POEA, or AMPA has a small toxic effect on embryonic cells alone at low levels, the combination of
two of them at the same final concentration is significantly deleterious (Figure 4).
Figure 4. Combined effects of G, AMPA, and POEA on three human cell types. The cells were incubated in serum-free medium for 24 h, and the products
were tested by pairs to a final concentration, where they are nontoxic alone on succinate dehydrogenase, of 0.05 (HUVEC) and 0.5% (293, JEG3). Results of
cellular death are evaluated through AK activity in relative units in comparison to nontreated cells (control = 1), and values are blank-subtracted (blank = no
AK); see the Materials and Methods. R360 and G are used as controls. SEs are shown in all instances (n = 16; **p < 0.01).
We have thus elucidated that R- and G-induced cell death can be due, at least
in part, to apoptosis via caspases 3/7 induction (Figure 5). The caspases are activated from 6 h with a maximum at 12 h in all cases, but umbilical primary
cells are 60−160 times more sensitive than lines (293 and JEG3, respectively) at this level. Moreover, G and R360 enhance exactly at the same concentration caspases, from 50 ppm (HUVEC). The adjuvants do not appear to be necessary
to render G as a death inducer at this level. Even G alone is 30% more potent
on this pathway than R. Surprisingly, G acted very rapidly at concentrations 500−1000 times lower than agricultural use on human cell apoptosis. This apoptotic pathway was also activated at levels 200 times lower for G on
caspases than its action on SD for umbilical cells, and for R at levels 60 times lower, in a four times shorter period (6−24 h). After 24 h of treatment, the
caspases returned to basal level when SD and AK react significantly. These data are consistent with a gradual loss of caspases 3/7 activity in apoptotic
cells that undergo secondary necrosis in vitro (44).
Figure 5. Time-dependent apoptosis through caspases 3/7 induction by R and
G in three human cell types. R360 and G, at similar concentrations and pH (as in Figure 1), were incubated for 6, 12, 18, or 24 h. The apoptotic pathway was
tested by the Caspase-Glo 3/7 assay, and results are presented in relative units to nontreated cells (control = 1). SEs are shown in all instances (n = 8).
Our results are confirmed by the morphology of the cells after treatment by R
(for instance R400, Figure 6B,D,E) in comparison with the normal cell types (A, C, F). Indeed, the very weak R concentration of 0.005% causes a very
important cell death, lack of adhesion, shrinking, and fragmentation in apoptotic bodies. This is confirmed in Figure 7 with the DNA fluorescent
labeling with DAPI, for example, with R360 at 0.5% over 24 h. The characteristic fluorescence of apoptotic cells evidencing DNA condensation is more visible with the herbicide than in controls (A, D, G) and more after R
treatment (C, F, I) than with G alone (B, E, H), for cell lines. The primary cells are similarly sensitive to G than to R, as for caspases activation in Figure 5.
Figure 6. Microphotographs of R-treated human cells. The cell types were without coloration (magnification, 100×; blue filter), HUVEC (A, B), 293 (C, D),
and JEG3 (E, F), and were incubated with 0.005% of R400 or not (controls) in serum-free medium for 24 h. Microphotographs were obtained with the Leïca
Microscopy Systems (model Leïca DC 100).
Figure 7. Increase of DNA condensation (DAPI test) in R360- or G-treated
human cells. The cell types HUVEC (A−C), 293 (D−F), and JEG3 (G−I) were incubated for 24 h with or without 0.5% R360 or G at equivalent concentrations. Staining of DNA with DAPI was examined with a microscope
model Leïca LMD 6000, using a fluorescent mode. Labeled DNA of viable cells was scattered throughout the nucleus, and bright condensation of chromatin
revealed apoptotic cells (magnification, 400×).
We had previously demonstrated (14) that G-based formulations were able to affect human placental cell viability at subagricultural doses (0.1% in 18 h)
and sexual steroid biosynthesis at lower nontoxic doses (0.01%) and that this was due at least in part to G, but its action was highly amplified by adjuvants,
the so-called inert ingredients of R formulations, kept confidential by the companies (9). However, the question of a specific cell line action or a time
reversible effect remained open. Benachour et al. (15) demonstrated that in embryonic cells as well as in normal human placenta and equine testis, there was a similar G-dependent endocrine disruption, through aromatase inhibition,
at nontoxic levels. The embryonic cells were even more sensitive: It was discovered that the cell mitochondrial activity was also reached in time- and
dose-dependent manners by the G formulation R360. The cytotoxicity was amplified around 14 times between 24 and 72 h (15), suggesting either a
bioaccumulation or a time-delayed effect and suggesting a cumulative impact, after endocrine disruption, of very low doses around G acceptable daily intake
(ADI: 0.3 mg/kg/j), according to the nature of the adjuvants.
To understand in vivo effects through the interpretation of the in cell impacts described above, it is necessary to have knowledge of the dilution and of the
processes leading to an elimination of the product in the body. This must be taken into account in regard to its bioaccumultation potential and time-delayed
effects. This is why we have measured the caspases activities at different times and G or R concentrations, after having previously demonstrated their
effects amplified with time within 3 days, on SD in embryonic and placental cells (15). Moreover, the metabolism of the herbicide has to be considered,
and the tests in this study of all the above-cited products approach this question.
All cell types, including primary cultures, react similarly at the membrane and
mitochondrial level, justifying the hypothesis that the cell lines used provide excellent models to study human cell toxicity, for instance in placental cells
(18). We show for the first time that embryonic and umbilical cells also have comparable sensitivity. The most reactive level reached appears to be the cell
membrane level for the different formulations, but not for G. The supposed “inert ingredients” play obviously and differently the role of cell membrane
disruptors, independently to G, as we have previously proposed (14), and this was suggested in fish, amphibians, and microorganisms (27, 45) or in plants (46). We now demonstrate that in human cells.
The second level is the mitochondrial membrane and the enzymatic reaction in it, SD, localized in the internal membrane in complex II of the respiratory
chain (47). It is altered in a comparable way, not proportional to G but relatively to the nature and the quantity of the adjuvants that we have previously listed (15). This means that the toxicity of G clearly varies with
formulations that must imperatively now be used in in vivo tests to study any toxicity (45); this also means that the ADI of G must take into account its
formulation, since 7.2 or 360 g/L of G may have comparable effects, considerably different to 400 g/L. It would even be more correct to use
precisely an ADI of R instead of G. It may also be time-dependent. These ideas are not taken into account yet for regulatory legislation.
The necessity to study combined effects also appears from our results. In fact,
the body is always exposed to mixtures and not to single compounds. We have previously demonstrated that mixtures could amplify toxicity for other widely
spread pollutants (2). For embryonic or neonatal cells, POEA, the major adjuvant, has the highest toxicity, either by itself or amplified 2−5 times in
combination with G or AMPA. It has already been shown that POEA is highly toxic for sea urchin embryos, impinging on transcription (28). It is also known
that in an aquatic environment, POEA has higher effects than R and G on bacteria, microalgae, protozoa, and crustaceans (12). In addition, the known
metabolism of G in the soil or plants is supposed to detoxify it in AMPA (11); however, here, we demonstrate that AMPA is more toxic than G in human
cells, especially on cell membrane. AMPA is also more stable in soil (48), in plants, and in food or feed residues (49), and more present in wastewater (2−35 ppm) than G [0.1−3 ppm; (50)]. It is not toxic alone at these
concentrations in our experiments, but it amplifies G or POEA toxicity in combination. The synergic toxicity of all of these compounds is now more
The induced resistance of placental cancer cells (51) could explain a specific difference for JEG3 cells at these levels. The placental cells could form an
efficient barrier to mixtures before their death, since the membranes are more resistant, and this could be due to the fact that there are carcinoma-derived
cells that have acquired a capacity to excrete xenobiotics.
The caspases 3/7 inducing apoptosis were in fact activated first within 6 h, and then, they decreased with cell mortality. This corroborates the timing observed
for another compound (36). The caspases induction by G alone is observed at doses that do not provoke cell or mitochondrial membrane damages, indicating
a clear G-apoptotic pathway always at subagricultural doses. Mixed with adjuvants, G in R formulations reached the other end points. This suggests
that the adjuvants could also play a role in total cell death, through necrosis characterized by organelle alterations with mitochondrial and cell membranes
swelling and ruptures (52). The most sensitive are umbilical HUVEC cells, for which apoptosis has been described (53-55), but very rarely induced by a
pesticide, for example, in the case of diallyl trisulfide (56). Surprisingly, this
phenomenon was observed for G and R at similar and low concentrations, as if a cell membrane death receptor was activated (57, 32), with no G penetration necessary. The modification of a dependency receptor is another pathway that
could be studied (58). The apoptotic cell appearance was microscopically confirmed. Then, our next step could be to study the necrotic/apoptotic ratio
In conclusion, mixtures called “formulations” change cell permeability, toxicity, and pathways of xenobiotics: In all cases, cell death is induced more by R than
by AMPA or G, and the latter provokes apoptosis (from 50 ppm in HUVEC cells) without membrane damage. By contrast, G mixed with adjuvants in R
formulations disrupts cell and mitochondrial membranes and promotes necrosis. It becomes obvious that the “threshold” level of action of the
herbicide should take into account the period and length of exposure, the presence of adjuvants, in particular POEA, metabolism, and bioaccumulation or
time-delayed effects. All of the above effects are demonstrated below the recommended herbicide agricultural dilutions (from 104 ppm). This clearly
confirms that the adjuvants in Roundup formulations are not inert. Moreover, the proprietary mixtures available on the market could cause cell damage and
even death around residual levels to be expected, especially in food and feed derived from R formulation-treated crops.
We thank CRIIGEN, Regional Council of Basse-Normandie, and Human Earth Foundation. This work was also supported by a grant from Fondation Denis
Guichard under the aegis of the Fondation de France. We declare that we have no competing financial interest. We thank Dr. Carine Travert for scientific revision of the manuscript.
This article references 58 other publications.
Feron, V. J., Cassee, F. R., Groten, J. P., van Vliet, P. W., and van Zorge,
J. A. 2002 International issues on human health effects of exposure to chemical mixtures Environ. Health Perspect. 110 893 899
Benachour, N., Moslemi, S., Sipahutar, H., and Séralini, G. E. 2007 Cytotoxic effects and aromatase inhibition by xenobiotic endocrine distrupters alone and in combination Toxicol. Appl. Pharmacol. 222 129 140
Tichy, M., Borek-Dohalsky, V., Rucki, M., Reitmajer, J., and Feltl, L. 2002 Risk assessment of mixtures: Possibility of prediction of interaction between
chemicals Int. Arch. Occup. Environ. Health 75 S133−S136
Monosson, E. 2005 Chemical mixtures: Considering the evolution of
toxicology and chemical assessment Environ. Health Perspect. 113 383 390
Acquavella, J. F., Bruce, H., Alexander, B. H., Mandel, J. S., Gustin, C.,
Baker, B., Champan, P., and Bleeke, M. 2004 Glyphosate biomonitoring for farmers and their families: Results from the farm family exposure study Environ. Health Perspect. 112 321 326
Takahashi, M., Horie, M., and Aoba, N. 2001 Analysis of glyphosate and its metabolite, aminomethylphosphonic acid, in agricultural products by HPLC
Clive, J. 2007 The global status of the commercialized
biotechnological/genetically modified crops: 2006 Tsitol. Genet. 41 10 12
IFEN 2006 Report on pesticides in waters: Data 2003−2004. Institut
Français de l’Environnement, Orleans, France Dossier 5 15 20
Cox, C. 2004 Glyphosate J. Pest. Reform. 24 10 15
Acquavella, J. F., Weber, J. A., Cullen, M. R., Cruz, O. A., Martens, M. A., Holden, L. R., Riordan, S., Thompson, M., and Farmer, D. 1999 Human ocular effects from self-reported exposures to Roundup herbicides Hum. Exp. Toxicol.
Williams, G. M., Kroes, R., and Munro, I. C. 2000 Safety evaluation and
risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for human Regul. Toxicol. Pharmacol. 31 117 65
Tsui, M. T., and Chu, L. M. 2003 Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of
environmental factors Chemosphere 52 1189 1197
Cox, C. 1998 Glyphosate (Roundup) J. Pest. Reform. 18 3 17
Richard, S., Moslemi, S., Sipahutar, H., Benachour, N., and Séralini, G. E.
2005 Differential effects of glyphosate and Roundup on human placental cells and aromatase Environ. Health Perspect. 113 716 720
Benachour, N., Sipahutar, H., Moslemi, S., Gasnier, C., Travert, C., and Séralini, G. E. 2007 Time and dose-dependent effects of Roundup on human
embryonic and placental cells and aromatase inhibition Arch. Environ. Contam. Toxicol. 53 126 133
Savitz, D., Arbuckle, T., Kaczor, D. M., and Curtis, K. 1997 Male pesticide
exposure and pregnancy outcome Am. J. Epidemiol. 146 1025 1036
Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., Van
der Saag, P. T., Van der Burg, B., and Gustafsson, J. A. 1998 Interaction of
estrogenic chemicals and phytoestrogens with estrogen receptor β Endocrinology 139 4252 4263
Letcher, R. J., Van Holstein, I., Drenth, H. J., Norstrom, R. J., Bergman,
A., Safe, S., Pieters, R., and van den Berg, M. 1999 Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental
JEG-3 and JAR choriocarcinoma cells Toxicol. Appl. Pharmacol. 160 10 20
L’Azou, B., Fernandez, P., Bareille, R., Beneteau, M., Bourget, C., Cambar,
J., and Bordenave, L. 2005 In vitro endothelial cell susceptibility to xenobiotics: Comparison of three cell types Cell Biol. Toxicol. 21 127 137
Thorin, E., and Shreeve, S. M. 1998 Heterogeneity of vascular endothelial cells in normal and disease states Pharmacol. Ther. 78 155 166
Peters, R. J. B. (2005) Man-Made Chemicals in Maternal and Cord Blood.
Annas, A., Granberg, A. L., and Brittebo, E. B. 2000 Differential response
of cultured human umbilical vein and artery endothelial cells to Ah receptor agonist treatment: CYP-dependent activation of food and environmental
mutagens Toxicol. Appl. Pharmacol. 169 94 101
Choi, W., Eum, S. Y., Lee, Y. W., Hennig, B., Robertson, L. W., and Toborek, M. 2003 PCB 104-induced proinflammatory reactions in human
vascular endothelial cells: Relationship to cancer metastasis and atherogenesis Toxicol. Sci. 75 47 56
Rathinasamy, K., and Panda, D. 2006 Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells FEBS J. 273 4114 4128
Kannan, K., Holcombe, R. F., Jain, S. K., Alvarez-Hernandez, X., Chervenak, R., Wolf, R. E., and Glass, J. 2000 Evidence for the induction of
apoptosis by endosulfan in a human T-cell leukemic line Mol. Cell. Biochem. 205 53 66
Peixoto, F. 2005 Comparative effects of the Roundup and glyphosate on mitochondrial oxidative phosphorylation Chemosphere 61 1115 1122
Marc, J., Mulner-Lorillon, O., Boulben, S., Hureau, D., Durand, G., and Bellé, R. 2002 Pesticide Roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation Chem. Res. Toxicol. 15 326 331
[ACS Full Text ACS Full Text], [PubMed], [ChemPort]
Marc, J., Le Breton, M., Cormier, P., Morales, J., Bellé, R., and Mulner-Lorillon, O. 2005 A glyphosate-based pesticide impinges on transcription
Bellé, R., Le Bouffant, R., Morales, J., Cosson, B., Cormier, P., and Mulner-
Lorillon, O. 2007 Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development J. Soc. Biol. 201 317 27
Le Bouffant, R., Cormier, P., Cueff, A., Bellé, R., and Mulner-Lorillon, O. 2007 Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint, DNA repair and apoptosis Cell. Mol. Life Sci.
Squirrell, D., and Murphy, J. (1997) Rapid detection of very low numbers of micro-organisms using adenylate kinase as a cell marker. A Practical Guide to Industrial Uses of ATP Luminescence in Rapid Microbiology, pp 107−113
Cara Technology Ltd., Lingfield, Surrey, United Kingdom.
Taatjes, D. J., Sobel, B. E., and Budd, R. C. 2008 Morphological and
cytochemical determination of cell death by apoptosis Histochem. Cell. Biol. 129 33 43
Scatena, R., Messana, I., Martorana, G. E., Gozzo, M. L., Lippa, S., Maccaglia, A., Bottoni, P., Vincenzoni, F., Nocca, G., Castagnola, M., and Giardina, B. 2004 Mitochondrial damage and metabolic compensatory
mechanisms induced by hyperoxia in the U-937 cell line J. Biochem. Mol. Biol. 37 454 459
Los, M., Wesselborg, S., and Schulze-Osthoff, K. 1999 The role of caspases in development, immunity, and apoptotic signal transduction:
Lessons from knockout mice Immunity 10 629 639
Omezzine, A., Chater, S., Mauduit, C., Florin, A., Tabone, E., Chuzel, F.,
Bars, R., and Benahmed, M. 2003 Long-term apoptotic cell death process with increased expression and activation of caspase-3 and -6 in adult rat germ cells
exposed in utero to flutamide Endocrinology 144 648 661
Liu, J. J., Wang, W., Dicker, D. T., and El-Deiry, W. S. 2005 Bioluminescent imaging of TRAIL-induced apoptosis through detection of
caspase activation following cleavage of DEVD-aminoluciferin Cancer Biol. Ther. 4 885 892
Galluzzi, L., Maiuri, M. C., Vitale, I., Zischka, H., Castedo, M., Zitvogel, L., and Kroemer, G. 2007 Cell death modalities: Classifications and
pathophysiological implications Cell Death Differ. 14 1237 43
Mosmann, T. 1983 Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays J. Immunol.
Denizot, F., and Lang, R. 1986 Rapid colorimetric assay for cell growth and
survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability J. Immunol. Methods 89 271 277
Crouch, S. P. M., Kozlowski, R., Slater, K. J., and Fletcher, J. 1993 The use
of ATP bioluminescence as a measure of cell proliferation and cytotoxicity J. Immunol. Methods 160 81 88
O’Brien, M., Moravec, R., Riss, T., and Promega Corporation (2003) Caspase-Glo 3/7 assay: Use fewer cells and spend less time with this
homogeneous assay. Cell Notes 6, 13−15.
Ren, Y. G., Wagner, K. W., Knee, D. A., Aza-Blanc, P., Nasoff, M., and Deveraux, Q. L. 2004 Differential regulation of the TRAIL death receptors DR4
and DR5 by the signal recognition particle Mol. Biol. Cell 15 5064 5074
Travert, C., Carreau, S., and Le Goff, D. 2006 Induction of apoptosis by
25-hydroxycholesterol in adult rat Leydig cells: Protective effect of 17beta-estradiol Reprod. Toxicol. 22 564 570
Riss, T. L., and Moravec, R. A. 2004 Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in
cell-based cytotoxicity assays Assay Drug Dev. Technol. 2 51 62
Cox, C., and Surgan, M. 2006 Unidentified inert ingredients in pesticides: Implications for human and environmental health Environ. Health Perspect.
Haefs, R., Schmitz-Eiberger, M., Mainx, H. G., Mittelstaedt, W., and Noga, G. 2002 Studies on a new group of biodegradable surfactants for glyphosate
Thomas, P. K., Cooper, J. M., King, R. H., Workman, J. M., Schapira, A. H.,
Goss-Sampson, M. A., and Muller, D. P. 1993 Myopathy in vitamin E deficient rats: Muscle fibre necrosis associated with disturbances of mitochondrial
Gard, J. K., Feng, P. C., and Hutton, W. C. 1997 Nuclear magnetic
resonance timecourse studies of glyphosate metabolism by microbial soil isolates Xenobiotica 27 633 644
Scalla, R. 1997 Sécurité alimentaire biotransformation des herbicides par
les plantes transgéniques résistantes. Oléagineux, Corps Gras, Lipides Dossier: Génie Génét. Oléagineux 4 113 119
Ghanem, A., Bados, P., Kerhoas, L., Dubroca, J., and Einhorn, J. 2007 Glyphosate and AMPA analysis in sewage sludge by LC-ESI-MS/MS after FMOC
derivatization on strong anion-exchange resin as solid support Anal. Chem. 79 3794 801
[ACS Full Text ACS Full Text], [PubMed], [ChemPort]
Xu, J., Peng, H., and Zhang, J. T. 2007 Human multidrug transporter ABCG2, a target for sensitizing drug resistance in cancer chemotherapy Curr.
Okada, H., and Mak, T. W. 2004 Pathways of apoptotic and non-apoptotic death in tumour cells Nat. Rev. Cancer 4 592 603
Solovyan, V. T., and Keski-Oja, J. 2005 Apoptosis of human endothelial cells is accompanied by proteolytic processing of latent TGF-beta binding
proteins and activation of TGF-beta Cell Death Differ. 12 815 826
Song, Y., Shen, K., and He, C. X. 2007 Construction of autocatalytic caspase-3 driven by amplified human telomerase reverse transcriptase
promoter and its enhanced efficacy of inducing apoptosis in human ovarian carcinoma Zhonghua Fu Chan Ke Za Zhi 42 617 622
Qiu, J., Gao, H. Q., Li, B. Y., and Shen, L. 2008 Proteomics investigation of protein expression changes in ouabain induced apoptosis in human umbilical
vein endothelial cells J. Cell. Biochem. 104 1054 1064
Xiao, D., Li, M., Herman-Antosiewicz, A., Antosiewicz, J., Xiao, H., Lew, K.
L., Zeng, Y., Marynowski, S. W., and Singh, S. V. 2006 Diallyl trisulfide inhibits angiogenic features of human umbilical vein endothelial cells by causing Akt
inactivation and down-regulation of VEGF and VEGF-R2 Nutr. Cancer 55 94 107
Ryu, H. Y., Emberley, J. K., Schlezinger, J. J., Allan, L. L., Na, S., and
Sherr, D. H. 2005 Environmental chemical-induced bone marrow B cell apoptosis: Death receptor-independent activation of a caspase-3 to caspase-8
Kroemer, G., Galluzzi, L., and Brenner, C. 2007 Mitochondrial membrane permeabilization in cell death Physiol. Rev. 87 99 163
Agents Classified by the IARC Monographs , Volumes 1–102 026148-68-5 A-alpha-C (2-Amino-9 H -pyrido[2,3- b ]indole) 025732-74-5 Acepyrene (3,4-dihydrocyclopenta[ cd ]pyrene) 3 Acetaldehyde associated with consumption of alcoholic 000103-90-2 Acetaminophen (see Paracetamol) (NB: Overall evaluation upgraded to Group 2A with supporting evidence from other relevant data) Acrylonitrile-
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