A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels.

Abstract

The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphosate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of surface waters. Roundup is being used increasingly in particular on genetically modified plants grown for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature rat fresh testicular cells from 1 to 10000ppm, thus from the range in some human urine and in environment to agricultural levels. We show that from 1 to 48h of Roundup exposure Leydig cells are damaged. Within 24-48h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to glyphosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and glyphosate (1ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus an endocrine impact at very low environmental doses, but only a high contamination appears to provoke an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested, and only with glyphosate in regulatory tests.

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A glyphosate-based herbicide induces necrosis and apoptosis in mature rat
testicular cells in vitro, and testosterone decrease at lower levels
Émilie Claira,b, Robin Mesnagea,b, Carine Traverta, Gilles-Éric Séralinia,b,⇑
aUniversité de Caen Basse-Normandie, EA2608, Institute of Biology, Esplanade de la Paix, 14032 Caen Cedex, France
bUniversité de Caen Basse-Normandie, Risk Pole MRSH-CNRS, and CRIIGEN, 40 rue de Monceau, 75008 Paris, France
a r t i c l ei n f o
Article history:
Received 23 June 2011
Accepted 9 December 2011
Available online xxxx
Keywords:
Roundup
Glyphosate
Testicular cells
Cytotoxicity
Endocrine disruption
a b s t r a c t
The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphos-
ate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of
surface waters. Roundup is being used increasingly in particular on genetically modified plants grown
for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature
rat fresh testicular cells from 1 to 10000 ppm, thus from the range in some human urine and in environ-
ment to agricultural levels. We show that from 1 to 48 h of Roundup exposure Leydig cells are damaged.
Within 24–48 h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to gly-
phosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses
in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and gly-
phosate (1 ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus
an endocrine impact at very low environmental doses, but only a high contamination appears to provoke
an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested,
and only with glyphosate in regulatory tests.
? 2011 Elsevier Ltd. All rights reserved.
1. Introduction
An environmentally-linked syndrome called testicular dysgene-
sis has emerged (Bay et al., 2006; Skakkebaek et al., 2001). It in-
cludes a decrease in sperm quantity and quality (Auger et al.,
1995; Carlsen et al., 1992), an increase in congenital malformations
such as cryptorchidism and hypospadias (Toppari et al., 2010), and
a preoccupying increase of testicular cancer incidence (Bergstrom
et al., 1996). This indicates that the testis is a sensitive target for
xenobiotics. The food/water/air intake of xenobiotics in the young
as well as in adults may lead to endocrine disruption at a reproduc-
tive and more specifically testicular level (Anway et al., 2006;
Savitz et al., 1997). In vitro, ex vivo and in vivo experiments are nec-
essary approaches to help us understand the mechanisms of xeno-
biotics actions at a developmental and/or adult stage.
In this work, we have chosen to test one of the most used pesti-
cides round the world. Roundup (R) formulations are non selective
herbicides composed of mixtures of glyphosate (G) and adjuvants
such as polyoxyethylene tallowamine (POEA) (Benachour et al.,
2007b). These compounds, with the G metabolite aminomethyl-
phosphonic acid (AMPA), are major contaminants in surface waters
withlevels reachingfor instance24 ppb forG in groundwater (IFEN,
2007). Moreover, these residues also concentrate in approximately
80% genetically modified plants grown for food and feed, which are
rendered R tolerant, up to 400 ppm (maximal residual levels, U.S.
EPA, 1998). We tested here R from 1 ppm to agricultural working
dilutions on rat testicular cells.
It is known that G is a weed killer inhibiting the shikimic acid
pathway in plants, essential for aromatic amino acids synthesis,
and it penetrates and is stabilized in the cells with the help of
the adjuvants (Cox, 1998, 2004). R used in our study contains
360 g/l of G and various xenobiotics added as adjuvants. Therefore
R is a good model to study in vitro combined effects, and especially
synergistic ones for xenobiotics (Benachour et al., 2007a; Richard
et al., 2005). In fact, G and/or R can also induce mortality in human
cells, revealed by disruptions of mitochondrial succinate dehydro-
genase, caspases 3/7, and adenylate kinase (Benachour and Séralin-
i, 2009). R is even responsible for oxidative damage in human
epidermal cells (Gehin et al., 2005). G and/or R also have side tar-
gets in mammals such as cytochrome P450 reductase, StAR, aroma-
tase and sexual steroid receptors of cells involved in reproduction
or in transfected human cells (Gasnier et al., 2009; Richard et al.,
2005; Stocco et al., 1995; Walsh et al., 2000).
In mammals, and rats in particular, the respiratory and hepatic
systems (Adam et al., 1997; Beuret et al., 2005) can be altered by
this herbicide, as well as hepatobiliary and reproductive functions
including sperm production or libido, and even fetal development
0887-2333/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2011.12.009
⇑Corresponding author at: Université de Caen Basse-Normandie, EA2608,
Institute of Biology, Esplanade de la Paix, 14032 Caen Cedex, France. Tel.: +33 (0)
2 31 56 54 89; fax: +33 (0) 2 31 56 53 20.
E-mail address: criigen@unicaen.fr (G-É. Séralini).
Toxicology in Vitro xxx (2012) xxx–xxx
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Toxicology in Vitro
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Page 2

(Chan and Mahler, 1992; Dallegrave et al., 2003, 2007; Yousef et al.,
1995). Therefore its impact in mammalian reproduction is docu-
mented, but not its direct possible testicular impact nor the mech-
anism of action or the sensitivity of adult gonadal cells.
R contamination may come from air (dermal or pulmonary dur-
ing spraying), water, feed and food. At present few studies have
been conducted to know the tissue concentration of G after expo-
sure and its possible bioaccumulation. However it was reported
that occupational exposure, primarily via the oral route, results
in urinary concentrations of G in the order of ppm (Acquavella
et al., 2004), in the range of our lower concentrations tested
(1 ppm of R corresponding to 0.36 ppm of G). More recently, it
was observed that after oral administration to rats of 10 ppm of
G, 30% were absorbed in males and 36% in females, with a peak ob-
served 2 h after administration. If the majority of G (90% after 72 h)
appears to be excreted via the feces or urine before metabolism,
this does not exclude the bioaccumulation in some tissues. One
percent of G persists after 7 days, located in particular in the colon
but also primarily in bone (Brewster et al., 1991). It is known that G
can bind to calcium ions, this occurs also in the soil (Sprankle et al.,
1975). More recently it has been shown that after oral ingestion of
10 ppm of the herbicide, it diffuses in mammalian tissues, with a
half-life of 15 h in rats, and G is then found in plasma at 5 ppm
(Anadon et al., 2009).
Typically these assays are performed after administration of a
single or a few doses of G in a short time, and this does not quantify
the bioaccumulation in the body of a long-term real environmental
exposure by air, water, food or feed, like through consumption of
Roundup-tolerant edible plants, such as most agricultural geneti-
cally modified organisms. In addition, only the active ingredient
in plants is well studied thus there is little information available
onits metabolites,andregarding
toxicokinetics.
To date, very few studies have been conducted on R effects on
primary cells; one study in particular was conducted by our group
on human umbilical cord cells (Benachour and Séralini, 2009),
where we demonstrated the necrotic and apoptotic capacities of
R at environmental levels. Consequently, in this work, we mea-
sured the differential specificities of R and G actions on adult rat
freshly separated testicular cells in order to know the threshold
of toxicity. These are Leydig, Sertoli, Sertoli and germ cells, and
germ cells alone. An increased mortality of these cells or a disrup-
tion in enzyme or hormone production could lead to a deleterious
effect on reproduction. Necrosis and apoptosis were assayed at
sub-agricultural dilutions of the herbicide R, and G, its compound
without adjuvants, and the endocrine disruption was tested at
non cytotoxic levels from 1 ppm. This work is the first study of
the side effects of the main herbicide of the world on primary tes-
ticular mammalian cells.
R adjuvants andtheir
2. Materials and methods
2.1. Animals
Healthy adult male albino Sprague–Dawley rats (70 days ± 5)
were obtained from Janvier (Le Genest-Saint-Isle, France) or from
the University Center of Biological Resources (Caen, France), and
were maintained on a 12 h light/dark cycle at 20–22 ?C. Standard
food and water were provided to the animals ad libitum.
2.2. Chemicals
Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham F12
were purchased from PAN (Biotech GmbH, Dutscher, Brumath,
France); and collagenase/dispase from Vibrio alginolyticus/Bacillus
polymyxa was from Roche (Mannheim, Germany). Soybean trypsin
inhibitor (STI), deoxyribonuclease I from bovine pancreas (DNAse
I), glyphosate (G) and serum replacement 3 were purchased from
Sigma–Aldrich (Saint-Quentin Fallavier, France). The 40,60-Di Ami-
dino-2-PhenylIndole (DAPI) was from Lonza (Verviers, Belgium).
Percoll was from GE Healthcare (Saclay, France). All other reagents
were of analytical grade. The herbicide R Bioforce?containing
360 g/l of acid G (R, homologation 9800036 corresponding to
100%) is a commercial formulation. Solutions of G (2% or 7.2 g/l
of G final) and R Bioforce?(diluted also to 2% final) were prepared
in DMEM/Ham F12 medium and adjusted to pH 7.4 and serially di-
luted in the same medium.
2.3. Isolation, purification and culture of Leydig cells
The rats were sacrificed and the testes were quickly decapsulat-
ed and placed in DMEM/Ham F12 nutrient medium (1:1, v/v). The
crude interstitial cells were separated from seminiferous tubules
by incubation in a medium containing collagenase/dispase
(0.05%), STI (0.005%), and DNase I (0.001%) at 32 ?C for 15 min in
a shaking water bath, followed by several decantations and a filtra-
tion through 30-mesh nylon. The Leydig cells were purified on a
discontinuous gradient of Percoll (20–80%) prepared in medium
as previously described (Lefevre et al., 1983). Leydig cell fractions
were collected, washed with the medium and their purity was
appreciated by histochemical staining for the specific 3b-hydroxy-
steroid dehydrogenase activity; 85–90% of positive cells were la-
beled. Leydig cells viability was determined by Trypan blue
exclusion test and was near 90%. After purification, Leydig cells
were maintained in DMEM/Ham F12 nutrient medium (1:1, v/v)
at 32 ?C (5% CO2, 95% air) with or without hCG, human homolog
of LH physiologically involved in endocrine regulation of Leydig
cells.
2.4. Isolation, purification and culture of Sertoli and germ cells
Sertoli and germ cells were isolated from the same rat testes by
three enzymatic digestions on pellets obtained after previously de-
scribed decantations. These pellets contain seminiferous tubules.
Briefly, after the first enzymatic digestion described above (32 ?C,
15 min), a second one was performed in the same conditions dur-
ing 30 min. The third and last one was in a solution with 0.1% hyal-
uronidase and 0.005% STI at 37 ?C for 30 min. After centrifugation
(900 rpm, 2 min), pellets contained Sertoli and germ cells. A second
centrifugation (2500 rpm, 10 min) was necessary to isolate germ
cells. Around 106germ cells/well were seeded in 96-wells plates
before treatments. The Sertoli and germ cells mixture was at a den-
sity of 2 ? 106cells/well in the same plates and cultured for 48 h in
Ham’s F12/DMEM medium (1:1, v/v) supplemented with serum
replacement 3 at 32 ?C (5% CO2and 95% air). On day 3, to obtain
purified Sertoli cells cultures when necessary, germ cells were re-
moved with an osmotic shock using a 20 mM Tris–HCl solution (pH
7.2). Treatments with different dilutions were applied on day 5 on
Sertoli cells.
2.5. Adenylate kinase measurement
The bioluminescent ToxiLightTMbioassay (Lonza, Verviers,
Belgium), developed by Crouch et al. (1993), is a non-destructive
enzymatic bioassay. It measures quantitatively the luminescence
of adenylate kinase (AK) of mammalian injured cells in culture
(Crouch et al., 1993). The AK is a membranous enzyme present in
all eukaryotic cells, and is released into culture medium when cells
are damaged (the membrane integrity is disrupted during necrosis
or secondary necrosis that occurs as a result of apoptosis). The AK
release in the medium converts ATP from ADP, which is then
2
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Please cite this article in press as: Clair, É., et al. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and
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Page 3

measured on a luminometer (Mithras LB 940, Berthold, Thoiry,
France); the bioluminescent reaction is produced by luciferase.
When the cytolysis increases, AK increases in the supernatants,
resulting in higher light intensity.
Before the assay, 105cells per well in 96-well plates (Dutsher,
Brumath, France) or 3 ? 105cells per well in 24-well plates
(Dutsher, Brumath, France) were treated with different dilutions
of R or G ± 1 UI/ml of hCG during 3, 6, 9, 12, 18, 24 or 48 h. The
adenylate kinase detection reagent (AKDR) was prepared in a buf-
fer (5 g/10 ml). Then 50 ll of supernatant were transferred to an
opaque black 96-well plate. Fifty microliter of AKDR reagent were
deposited into each well. The plates were then placed under agita-
tion for 15 min in the dark, and light was measured using the
luminometer.
2.6. Caspase 3/7 activity
The Caspase-GloTM3/7 assay (Promega, Paris, France) measures
the activities, in 96-well white plates (GBO, Dutscher, France), of
caspases 3 and 7, key-caspases of apoptosis, in cell cultures using
a bioluminescence-based method. The reagent contained a pro-
luminescent substrate of caspases 3/7, containing the tetrapeptide
sequence Z-DEVD-aminoluciferin, in a buffer including a detergent
for cell lysis and other components stabilizing caspases activities
(Bondzio et al., 2008; Liu et al., 2005; Riss and Moravec, 2004).
After cell lysis, the cleavage of the substrate by caspases released
aminoluciferin, which was then able to generate a ‘‘glow-type’’
luminescence, produced by luciferase related to the caspase 3/7
activity in the sample. This assay was designed for automated
high-throughput screening of caspases 3/7 activities, specific for
apoptosis.
Before the assay, 105cells per well in 96-well white transparent
background plate were treated with different dilution of R or
G ± 1 UI/ml of hCG during 3, 6, 9, 12, 18, 24 or 48 h. The Caspase-
Glo?3/7 reagent was prepared in the buffer provided (Promega,
Paris, France). After 30 min at room temperature, 50 ll of Cas-
pase-Glo?3/7 reagent was added to 50 ll of culture medium con-
taining the cells previously treated in each well. After shaking the
plate during 15 min, an incubation period of 45 min at room tem-
perature in the dark was needed to stabilize the signal before lumi-
nescence measurement with the luminometer.
2.7. Analysis of DNA in situ by DAPI
DAPI is a fluorescent stain that binds strongly to DNA after pass-
ing through cell membrane. After a 24 h incubation in presence of
various dilutions of G or R, 24-wells plates were centrifuged
(900 rpm, 15 min) and the medium was removed slowly. Leydig
cells (3 ? 105/well) were fixed for a day in absolute ethanol–
chloroform–acetic acid (6:3:1, v/v/v) at ?20 ?C. The wells were
washed with PBS (pH 7.4) and incubated with 1 lg/ml of a solution
containing DAPI during 30 min (Travert et al., 2006). Each well was
washed with water and then examined with a microscope using a
fluorescent mode (DMLB, Leica). Labeled DNA of viable cells was
scattered throughout the nucleus, and bright condensation of chro-
matin revealed apoptotic cells (magnification 400?).
2.8. 3b-HSD activity
Leydig cells, previously prepared and seeded in 96-well plates
as described above, were exposed for 24 h to different concentra-
tions of R Bioforce?, or equivalent non cytotoxic concentrations
of G, in medium DMEM/HamF12 to 32 ?C (5% CO2, 95% air). The
3b-hydroxysteroid dehydrogenase (3b-HSD), a Leydig cell specific
enzyme involved in particular in testosterone synthesis, was mea-
sured at the end of the treatment. The 3b-HSD reagent containing
DHEA (substrate), NAD (cofactor), NBT and nicotinamide was
added to wells containing Leydig cells pretreated and then incu-
bated at 37 ?C for 45–60 min. Once the cells are stained brown, a
solution of acetic acid (10%) was added to each well to solubilize
formazan crystals previously formed. The 3b-HSD enzyme activity
was then evaluated by reading the optical density of each well at
560 nm (formazan) through a plate reader (Mithras LB 940,
Berthold, Thoiry, France).
2.9. Radioimmunoassay (RIA) of testosterone
The radioimmunoassay was performed on the same Leydig cells
by competition and stopped using the method of activated char-
coal. Indeed, the steroid dose is in competition with its tritiated
counterpart by incubating 200 ll of standard solution of unlabeled
testosterone (7.5–800 pg of testosterone/200 ll phosphate buffer),
phosphate buffer (0.1 M Na2HPO4, 0.9% NaCl, 0.5% BSA, 0.01% NaN3
– pH 7.4) or culture supernatant with 100 ll of radioactive testos-
terone (3000 cpm/100 ll phosphate buffer) and 100 ll of rabbit
anti-testosterone antibody (final concentration 1/18000). After
30 min at room temperature, the mixture is placed at 4 ?C until
the next day. Then 500 ll of charcoal/dextran (50%/5%) at 4 ?C
are added. After 10 min of incubation at 4 ?C, the tubes were cen-
trifuged 10 min at 2400 rpm at 4 ?C and the radioactivity of the
supernatant was then counted. The sensitivity of the assay was
12 pg of testosterone per tube.
2.10. Measurement of mRNA expression of aromatase, androgen
receptor and estrogen receptor a and b by real-time PCR
After exposure of Leydig cells for 24 h at different non cytotoxic
concentrations of R or G in 6-well plates cell pellets were recovered
and placed in the presence of Trizol to degrade the cells. Then chlo-
roform was added to recover the aqueous phase containing the
RNA. Precipitation of RNA is done by adding isopropanol and wash-
ing by adding ethanol (70%). After a denaturation step (10 min at
55–60 ?C), the integrity of total RNA was controlled by dosing
(260–280 nm) and by electrophoresis on agarose gel (1.5%) labeled
by bromide ethidium. To achieve the reverse transcription (RT)
250 ng of RNA were used and placed in the presence of 200 U of
MMLV-RT (Moloney murine leukemia virus reverse transcriptase),
0.2 g of random primers, 500 mM of each dNTP and 20 U of recom-
binant RNasin?. The samples were then placed 90 min at 37 ?C to
obtain the cDNA, the reaction was stopped by 5 min at 75 ?C. The
polymerase chain reaction was performed on cDNA using the
method GoTaq?qPCR Master Mix (Promega). The PCR primers
used are: L19 50-GGA ATC TAA GAA GAT TGA CCG TC-30and
30-GCC TTG TCT GCC TTC AGT TT-50; aromatase 50- CGT CAT GTT
GCT TCT CAT CG-30and 30-TAC CGC AGG CTC TCG TTA AT-50; estro-
gen a receptor 50-AAT TCT GAC AAT CGA CGC CAG-30and 30-GTG
CTT CAA CAT TCT CCC TCC TC-50; estrogen b receptor 50-CTT GCC
CAC TTG GAA ACA TC-30and 30-CCA AAG GTT GAT TTT ATG GCC-
50; androgen receptor 50-TGG GGA CAT GCG TTT GGA CAG T-30
and 30-GCT GCC ACA AGT GAG AGC TCC G-50. The PCR conditions
were an initial step at 95 ?C for 3 min, then 40 cycles of 30 s at
95 ?C and 60 ?C for 60 s. mRNA levels of aromatase, estrogen recep-
tor a and b and androgen receptor were normalized using the L19
control gene.
2.11. Statistical analysis
All data are presented as the means ± Standard Errors (SEM).
The experiments were repeated in triplicates on different months
from three independent cultures each time (n = 9) unless other-
wise specified. To compare the results, statistically significant dif-
ferences from controls were determined by an Anova test followed
É. Clair et al./Toxicology in Vitro xxx (2012) xxx–xxx
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Page 4

by Bonferroni post-test with p < 0.001 (⁄⁄⁄⁄), p < 0.005 (⁄⁄⁄), p < 0.01
(⁄⁄) and p < 0.05 (⁄).
3. Results
Cytotoxicities of G alone and with adjuvants (R), from 50 ppm to
agricultural working dilutions (200 times more), were simulta-
neously measured on 70-day isolated adult rat testicular cells: Ley-
dig alone, Sertoli with germ cells, purified Sertoli, and finally germ
cells alone. Significant membrane degradations were provoked by
RinLeydigcellswithin24 hfrom0.1%(1000 ppm),thusatrelatively
high levels, however 10 times below the lowest agricultural dilu-
tions (Fig. 1a). This tendency was visible very rapidly from 1 h of
treatment and was persistent (Fig. 2). This phenomenon increases
up to five times more than in controls at higher doses; only at these
doses Leydig cells reacted as though they were around 50% more
resistant after 48 h of treatment (Fig. 1b). Moreover, isolated Sertoli
cells were also sensitive (but maximally two times over controls)
from0.05%(500 ppm),in24 h(Fig.1e).BycontrastSertolicellswere
almostinsensitiveto herbicide-inducedmortalityinthepresenceof
germcells,likegermcellsalone(Fig.1c,d,g,h).Thisdoesnotexclude
otherlimitedlightphenomenaathigherdoses(Fig.1g). Thekinetics
of membranedegradation was studied in Leydig cells, which are the
mostsensitivetoR(Fig.2).Rcytotoxicitywasobservedalreadyafter
1 h, and was maximal from then on and up to 24 h. We confirmed
that G alone had no action at any time over 24 h in Leydig cells.
Fig. 1. Effects of Roundup or Glyphosate (empty diamonds) in DMEM/Ham F12 medium after 24 h (left column) or 48 h (right) on adenylate kinase activities of Leydig (a and
b), Sertoli with germ cells (c and d), purified Sertoli (e and f) and germ (g and h) cells. Cytotoxicity of Roundup Bioforce?and glyphosate alone were measured through
adenylate kinase activities indicating membrane degradations in primary cultures of testicular cells. The cells were exposed to different doses of Roundup Bioforce?or
equivalent doses of glyphosate in DMEM/HamF12 medium in 96-wells plate at 32 ?C (5% CO2, 95% air) during 24 h or 48 h. Roundup and glyphosate were used at similar pH.
All studies for each concentration were repeated three times and in three different experiments. Results of cell death are presented in Relative Luminescence Units (R.L.U.)
compared to controls. SEMs are shown in all instances (Anova test p < 0.001⁄⁄⁄⁄; p < 0.005⁄⁄⁄; p < 0.01⁄⁄and p < 0.05⁄).
4
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Page 5

Signs of mortality were also observed through caspases 3/7
activity reduction induced by R. This was visible in all cells in very
similar profiles approximately from 0.1% (1000 ppm) with a max-
imal effect in 24–48 h (Fig. 3). These effects were comparable in
Leydig cells for membrane degradations, and indicate clearly a ma-
jor necrosis. However, G alone induced apoptosis in 48 h from 0.5%
(5000 ppm) in isolated germ cells and in Sertoli and germ cells
mixture (Fig. 3d, h). A minor apoptosis induction was also observed
in Leydig cells from 1000 ppm. The results of kinetic study of cas-
pase 3/7 activities on Leydig cells were fully consistent with those
obtained in Fig. 2: the decrease of these activities with R was
amplified between 1 and 24 h starting from 0.1% (1000 ppm)
(Fig. 4), reaching rapidly its maximum (after 6 h). Moreover, after
6 h of treatment, R first then G induced a punctual apoptosis phe-
nomenon in Leydig cells. The nuclei aspects in these cells (Fig. 5)
showed clear chromatin compaction after 24 h for R (1%). This is
characteristic of programmed cell death. For G alone (1%) or lower
R concentrations (0.05%) on Leydig cells, always in 24 h, an in-
crease in DNA compaction was observed. In Leydig cells, mem-
brane degradation and caspases 3/7 measurements within 24 h
were also measured in presence or absence of hCG, the human
homolog of LH (Fig. 6) that physiologically stimulates steroidogen-
esis. The cytotoxicity of treatments on Leydig cells are thus con-
firmed in presence of hCG, but are slightly reduced or delayed.
Concerning endocrine disruption study, R and G had no impact at
non-cytotoxic doses on 3b-HSD activity in these cells (Fig. 7).
However, testosterone production measured by radioimmunoas-
say was inhibited at 1 ppm by G and R (Fig. 8). This was not the
case on the androgen and estrogen receptors (a and b) mRNA lev-
els (Fig. 9b–d). By contrast G increases significantly but punctually
(and R with a light tendency) the aromatase mRNA (Fig. 9a), at
10 ppm.
4. Discussion
We developed here a model to investigate the effects of xenobi-
otics on mammalian reproductive cells, and especially testicular
ones, at different environmental levels. We know that the testis
is a sensitive target (Toppari et al., 2010). The originality of this
work lies in the study of the same chemicals on all main testicular
cell types simultaneously: Leydig, and Sertoli cells exposed in asso-
ciation or not with germ cells.
4.1. Leydig cells
Rat Leydig cells have been already proven to be very suitable
for assessing the toxicity and hormonal activities of xenobiotics
(Akingbemi et al., 2004). As a matter of fact, in this experiment,
Leydig cells seem to be the most differentially sensitive to R by
contrast to G, by the amplitude of the effect. There was only a very
light action of G on caspases 3/7 activities after 48 h. The impor-
tant R impact versus G was already observed by our group for var-
ious cell lines or fresh cells, for instance in human placenta and
Fig. 2. Kinetics of cellular death revealed by membrane degradation in Leydig cells after 1–24 h treatments by Roundup or glyphosate alone (empty diamonds). Cytotoxicity
of Roundup Bioforce?and glyphosate alone were measured through adenylate kinase activities indicating membrane degradations like previously. For more details see the
caption for Fig. 1.
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umbilical cord, embryonic kidney and liver (Benachour and
Séralini, 2009; Benachour et al., 2007b; Gasnier et al., 2009,
2010, 2011; Richard et al., 2005). In other works for instance, R
is also inducing toxicity on human epidermal cell lines (Gehin
et al., 2005). Since in Leydig cells the membrane degradations
were visible from 1 h with R, direct phenomena can occur at this
level. Here we demonstrated a cytotoxic effect of R by membrane
degradation at doses 1000 times lower than the commercial G-
based formulation, or as underlined, 10 times under the lowest
agricultural dilutions and around eight times less than the maxi-
mal level of residues authorized in GM feed. R disrupts the mam-
malian cell membrane and not G most probably via incorporation
of adjuvants such as POEA at this level.
However after a longer exposure of Leydig cells (48 h), if the
threshold of sensitivity was unchanged, the amplitude of the cell
membrane degradation was divided by approximately 2. We could
hypothesize a reduction of the membrane fluidity due to the inter-
action of adjuvants (Riechers et al., 1994), a new expression of
xenobiotics excretion proteins (Melaine et al., 2002), or directly
on a reduction of AK bioavailability.
Modification of membrane fluidity and/or disruption of mem-
brane potential can induce cell death by apoptosis, because a de-
creased membrane potential and the pore openings can allow the
release of cytochrome c and APAF-1. This can cause apoptosis via
the mitochondrial pathway dependent on caspases (Sarda-Mantel
et al., 2004; Vilches Troya, 2005).
Fig. 3. Effects of Roundup or Glyphosate (empty diamonds) after 24 h (left column) or 48 h (right) on caspases 3/7 activities of Leydig (a and b), Sertoli with germ cells (c and
d), purified Sertoli (e and f) and germ (g and h) cells. The effects were evaluated by the Caspases 3/7?assay. All conditions are similar as previously (see Fig. 1 legend); and
studies for each concentration were repeated two or three times in three different experiments. The results are presented in relative luminescence unit and compared to non-
treated cells (control = 1). SEMs are also shown in all instances (Anova test p < 0.001⁄⁄⁄⁄; p < 0.005⁄⁄⁄; p < 0.01⁄⁄and p < 0.05⁄).
6
É. Clair et al./Toxicology in Vitro xxx (2012) xxx–xxx
Please cite this article in press as: Clair, É., et al. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and
testosterone decrease at lower levels. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2011.12.009

Page 7

We then tested if the cytotoxicity of R measured through mem-
brane degradation was mostly due to necrosis, or apoptosis imply-
ing often a secondary necrosis. Necrosis is characterized by an
inflammatory reaction, i.e. swelling and bursting of organelles
and cells leading to a significant membrane rupture and by a re-
lease of the contents of their cytoplasm (Leist and Jaattela, 2001;
Sarda-Mantel et al., 2004). Apoptosis is characterized by a nuclear
condensation before the nuclear fragmentation, DNA degradation
and changes in mitochondria, activation of caspases (and especially
caspase 3) as well as cytoplasmic condensation, chromatin com-
paction and crush of the previous cell into apoptotic bodies, subse-
quently destroyed (Gerschenson and Rotello, 1992; Sarda-Mantel
et al., 2004; Vilches Troya, 2005). Apoptosis occurs either by an
extrinsic or intrinsic pathway. The extrinsic pathway is provoked
through stimulation of a plasma membrane death receptor (for in-
stance with a ligand such as a hormone, growth factor, cytokine or
a toxin). The intrinsic pathway corresponds to the release of mito-
chondrial signals such as cell stress caused by DNA damage, heat
shock, cell suicide, lack of nutrients (Brune, 2003; Csipo et al.,
1998; Popov et al., 2002). These signals might accumulate during
cell exposure to xenobiotics that can induce stress leading to
apoptosis.
The caspases 3/7 activities as apoptosis indicator collapsed after
1 h in Leydig cells, with no increase at all (except a very light one
after 6 h) but a decrease; this indicates no significant apoptosis in
comparison to the basal activity in controls. We deduced that the
cellular death was mostly due to necrosis, overall after adenylate
kinase release, without excluding a light apoptosis visible after
24–48 h of G treatment. At this time, most necrotic degraded cells
were not visible anymore (Fig. 5).
Leydig cells under hormonal treatment (hCG; Fig. 6), like in an
in vivo context, were still reactive to R. This stimulation of steroid
synthesis by the LH substitute had even a slightly protecting effect
from necrosis on these cells; it induces vitality as observed in other
cases (Nagai, 1992). Hormones are important for survival and
physiological function of testicular cells. Endocrine disruption
was then studied on mRNA levels for aromatase, androgen and
estrogen (a and b) receptors, 3b-HSD activity and testosterone pro-
duction at non cytotoxic doses, i.e. environmental ones, also found
in urine agricultural workers and their families (1 ppm). In this
work, the only endocrine effects in our conditions were a testoster-
one decrease by R and G and an aromatase mRNA increase by G.
The latter may be secondary to an inhibition of this enzymatic
activity previously shown, due to a direct interaction of G in the
aromatase active site (Richard et al., 2005). The testosterone pro-
duction inhibition and the consecutive recovery may be due to
an inhibition in the early steroidogenic pathway for instance on
StAR but not on 3b-HSD activity (Walsh et al., 2000).
An endocrine disruption in testicular cells could result in ad-
verse effects on the reproductive system including epigenetics
ones in offsprings (Anway et al., 2005), and more importantly it
could provoke a decrease in sperm count and sperm production
during adulthood, a decrease in the serum testosterone level at
puberty and an increase of abnormal sperms in rats (Dallegrave
Fig. 4. Kinetics of caspases 3/7 activities in Leydig cells after 1–24 h of treatments by Roundup or glyphosate alone (empty diamonds). Cytotoxicities of Roundup Bioforce?
and glyphosate alone were measured through caspases 3/7 activities like previously for all other details (see the caption for Fig. 1). All studies for each concentration were
repeated two times on three manipulations.
É. Clair et al./Toxicology in Vitro xxx (2012) xxx–xxx
7
Please cite this article in press as: Clair, É., et al. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and
testosterone decrease at lower levels. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2011.12.009

Page 8

et al., 2007). After exposure to G, various effects were also observed
in male rabbit reproductive health like a reduced body weight, li-
bido, ejaculate volume, sperm concentration, osmolarity of semen,
and an increase of abnormal or dead sperm (Yousef et al., 1995).
4.2. Sertoli cells, germ cells alone and Sertoli/germ cells co-cultures
R also induces a caspases 3/7 collapse for all cells, indicating a
necrosis with a smaller membrane degradation than with Leydig
cells, and mostly in 24 h. This is again in contrast to G impact alone,
which corresponds to a clear caspases induction in germ cells and
Sertoli/germ cells associations. This appears to be due to germ cells
sensitivity, since Sertoli cells are not significantly reactive. More-
over G could penetrate more easily in germ cells due to membrane
specificities. We know that G alone may penetrate into cells
(Gasnier et al., 2011). Whether it corresponds to an in vitro artifact
on isolated cells which have to be checked in in vivo experiments.
However, it is well known that germ cells do undergo significant
apoptosis during their differentiation (Petre-Lazar et al., 2007),
which could well be amplified by environmental stressors (Anway
et al., 2006). It has been observed that rats fed with R-treated
transgenic plants undergo ultrastructural disorders in their Sertoli
cells, such as transcription disruptions, nuclear and reticulum
changes, possibly due to herbicide residues (Vecchio et al., 2004).
Surprisingly, while germ cells have less transporters to mediate
cellular efflux of xenobiotics than Sertoli or Leydig cells (Melaine
et al., 2002; Tribull et al., 2003), they were at a membrane degra-
dation level insensitive to G. It was the same in Sertoli associated
Fig. 5. Study of chromatin condensation by DAPI-labeling (a–d) and cellular aspect (e and f) on primary Leydig cells cultures in DMEM/Ham F12 medium after 24 h of
treatment by Roundup or glyphosate alone. Bright condensation of chromatin revealed apoptotic cells (magnification 400?). Yellow arrow shows normal chromatin
condensation and the blue arrow evidences compacted chromatin. DAPI labeling in (a) control cells, (b) cells treated with glyphosate 1%, (c) Roundup 0.05%, (d) Roundup 1%;
(e) control unstained cells and (f) with Roundup 1%.
8
É. Clair et al./Toxicology in Vitro xxx (2012) xxx–xxx
Please cite this article in press as: Clair, É., et al. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and
testosterone decrease at lower levels. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2011.12.009

Page 9

to germ cells; their association was protective, as shown in other
studies (Benbrahim-Tallaa et al., 2002).
In conclusion, the general sensitivity of all cells to R either
through direct membrane degradation or caspases 3/7 disruptions
can be explained either by a combined sensitivity to G and adju-
vants forming R, or to a major sensitivity to some adjuvant(s)
alone. Previous studies on human umbilical cord fresh cells, and
cell lines from embryo and choriocarcinoma indicate in fact a great
sensitivity to POEA alone first, the main adjuvant; but this also did
not exclude a combined effect with G. In vivo studies on rats (Adam
et al., 1997) reached similar conclusions.
From 24 h R always induced necrosis in all testicular cells, and
only germ cells and to a lesser extent Leydig cells were affected by
apoptosis after 48 h of G alone. The differences in structures of cell
membranes of the three testicular cell types used for this work
may explain the differential effects of G and R on cells by differen-
tial membrane composition, which impacts fluidity and membrane
resistance. Moreover it has been shown previously that a chemical
can induce apoptosis or necrosis depending on the applied dose be-
cause of bioaccumulation, genomic effect or oxidative stress (Kan-
duc et al., 2002; Uezono et al., 2001). It is also known that R and G
cause a dose dependent increase in sister chromatid exchange in
human cells, with a greater effect of R (Bolognesi et al., 1997). In
addition, we should note that a recent study reports that AMPA,
the main metabolite of G (in R) is clastogenic on human cells
in vitro and genotoxic both in vitro and in vivo (Manas et al.,
2009). As previously described, these DNA disruptions like the ones
observed by nuclear shrinkage in our work could lead to necrosis.
People working with herbicides are typically exposed for much
longer and repeatedly to the tested doses, however this has to be
confirmed in vivo.
Taken together, the in vitro disturbances by R on testosterone
and aromatase in rat testicular cells around 1–10 ppm at non toxic
levels, underline the environmental endocrine disruptions possible
by G-based herbicides. Moreover Leydig cells are exposed to this
kind of environmental doses (Acquavella et al., 2004) because
1 ppm was found in human urine and thus was present in blood.
When 10 ppm of G are given to rats, half was still found in plasma
15 h later (Anadon et al., 2009). Moreover, in testis, Leydig cells and
blood vessels are in fact very close.
We know in addition that pesticide residues may bioaccumu-
late because of adjuvants. In our works, the presence of adjuvants
in commercial formulations is decisive in inducing most herbicide
side effects; this is why the present regulatory in vivo tests with G
alone to study chronic toxicity are hardly relevant. It is thus neces-
sary to revise the safety of formulations (Mesnage et al., 2010;
Monosson, 2005; Tichy et al., 2002). Our studies also represent an-
other method of investigation for understanding the possible ef-
fects of xenobiotics on the decline of male reproductive functions.
Fig. 6. Effects of Roundup or Glyphosate alone (empty diamonds) in medium complemented or not with hCG (larger symbols) after 24 h of treatment on Leydig cells.
Cytotoxicites of Roundup Bioforce?and glyphosate alone were evaluated on primary cultures of Leydig cells through adenylate kinase (a) and caspases 3/7 (b) activities as
previously described (see caption for Figs. 1 and 3). All studies for different concentrations were repeated three times and in three different experiments. SEMs are also
indicated.
Fig. 7. Effects of Roundup Bioforce?or Glyphosate in DMEM/Ham F12 medium
after 24 h of treatment on 3b-HSD Leydig primary cells activity in culture cells. The
cells were exposed to different doses of Roundup Bioforce?or equivalent non-
cytotoxic doses of glyphosate in DMEM/HamF12 medium in 96-wells plate at 32 ?C
(5% CO2, 95% air) during 24 h. Roundup and glyphosate were used at similar pH. All
studies for each concentration were repeated three times and for three different
manipulations. SEMs are shown in all instances (Anova test p < 0.001⁄⁄⁄; p < 0.01⁄⁄
and p < 0.05⁄).
Fig. 8. Effects of Roundup Bioforce?and glyphosate on the production of testos-
terone by Leydig cells in primary cultures after 24 h of exposure to various non-
cytotoxic concentrations. Changes in production of testosterone secreted into the
culture medium of primary Leydig cells previously prepared and incubated at 32 ?C
in the presence or absence of xenobiotics was measured using the radioimmuno-
assay method. All operations for each concentration were repeated three times on
three different manipulations. The SEMs are indicated in the figure (Anova test
p < 0.001⁄⁄⁄⁄; p < 0.005⁄⁄⁄; p < 0.01 and⁄⁄p < 0.05⁄).
É. Clair et al./Toxicology in Vitro xxx (2012) xxx–xxx
9
Please cite this article in press as: Clair, É., et al. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and
testosterone decrease at lower levels. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2011.12.009

Page 10

Conflict of interest statement
The authors declare that they have no conflict of interest.
Acknowledgments
We thank C. Edine and S. Edy for their technical assistance dur-
ing Leydig cells isolation and F. Hilary for secretarial assistance. For
financial support and fellowships (to E.C. and R.M.) we would like
to thank CRIIGEN (Committee for Independent Research and
Information on Genetic Engineering), and the Regional Council of
Basse-Normandie. The association CERES and Foundation Charles
Leopold Mayer are gratefully acknowledged for their support in
these experiments. We thank Herrade Hemmerdinger for proof-
reading the English version of the manuscript.
References
Acquavella, J.F., Alexander, B.H., Mandel, J.S., Gustin, C., Baker, B., Chapman, P.,
Bleeke, M., 2004. Glyphosate biomonitoring for farmers and their families:
results from the Farm Family Exposure Study. Environ. Health Perspect. 112,
321–326.
Adam, A., Marzuki, A., Abdul Rahman, H., Abdul Aziz, M., 1997. The oral and
intratracheal toxicities of ROUNDUP and its components to rats. Vet. Hum.
Toxicol. 39, 147–151.
Akingbemi, B.T., Sottas, C.M., Koulova, A.I., Klinefelter, G.R., Hardy, M.P., 2004.
Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is
associated with reduced pituitary luteinizing hormone secretion and decreased
steroidogenic enzyme gene expression in rat Leydig cells. Endocrinology 145,
592–603.
Anadon, A., Martinez-Larranaga, M.R., Martinez, M.A., Castellano, V.J., Martinez, M.,
Martin, M.T., Nozal, M.J., Bernal, J.L., 2009. Toxicokinetics of glyphosate and its
metabolite aminomethyl phosphonic acid in rats. Toxicol. Lett. 190, 91–95.
Anway, M.D.,Cupp,A.S., Uzumcu,
transgenerational actions of endocrine disruptors and male fertility. Science
308, 1466–1469.
M., Skinner,M.K., 2005.Epigenetic
Anway, M.D., Memon, M.A., Uzumcu, M., Skinner, M.K., 2006. Transgenerational
effect of the endocrine disruptor vinclozolin on male spermatogenesis. J. Androl.
27, 868–879.
Auger, J., Kunstmann, J.M., Czyglik, F., Jouannet, P., 1995. Decline in semen quality
among fertile men in Paris during the past 20 years. N. Engl. J. Med. 332, 281–
285.
Bay, K., Asklund, C., Skakkebaek, N.E., Andersson, A.M., 2006. Testicular dysgenesis
syndrome: possible role of endocrine disrupters. Best Pract. Res. Clin.
Endocrinol. Metab. 20, 77–90.
Benachour, N., Moslemi, S., Sipahutar, H., Seralini, G.E., 2007a. Cytotoxic effects and
aromatase inhibition by xenobiotic endocrine disrupters alone and in
combination. Toxicol. Appl. Pharmacol. 222, 129–140.
Benachour, N., Séralini, G.E., 2009. Glyphosate formulations induce apoptosis and
necrosis in human umbilical, embryonic, and placental cells. Chem. Res. Toxicol.
22, 97–105.
Benachour, N., Sipahutar, H., Moslemi, S., Gasnier, C., Travert, C., Seralini, G.E.,
2007b. Time- and dose-dependent effects of roundup on human embryonic and
placental cells. Arch. Environ. Contam. Toxicol. 53, 126–133.
Benbrahim-Tallaa, L., Tabone, E., Tosser-Klopp, G., Hatey, F., Benahmed, M., 2002.
Glutathione S-transferase alpha expressed in porcine Sertoli cells is under the
control of follicle-stimulating hormone and testosterone. Biol. Reprod. 66,
1734–1742.
Bergstrom, R., Adami, H.O., Mohner, M., Zatonski, W., Storm, H., Ekbom, A., Tretli, S.,
Teppo, L., Akre, O., Hakulinen, T., 1996. Increase in testicular cancer incidence in
six European countries: a birth cohort phenomenon. J. Natl. Cancer Inst. 88,
727–733.
Beuret, C.J., Zirulnik, F., Gimenez, M.S., 2005. Effect of the herbicide glyphosate on
liver lipoperoxidation in pregnant rats and their fetuses. Reprod. Toxicol. 19,
501–504.
Bolognesi, C., Bonatti, S., Degan, P., Gallerani, E., Peluso, M., Rabboni, R., Roggieri, P.,
Abbondandolo, A., 1997. Genotoxic activity of glyphosate and its technical
formulation Roundup. J. Agric. Food Chem. 45, 1957–1962.
Bondzio, A., Stumpff, F., Schon, J., Martens, H., Einspanier, R., 2008. Impact of Bacillus
thuringiensis toxin Cry1Ab on rumen epithelial cells (REC) – a new in vitro
model for safety assessment of recombinant food compounds. Food Chem.
Toxicol. 46, 1976–1984.
Brewster, D.W., Warren, J., Hopkins 2nd, W.E., 1991. Metabolism of glyphosate in
Sprague–Dawley rats: tissue distribution, identification, and quantitation of
glyphosate-derived materials following a single oral dose. Fundam. Appl.
Toxicol. 17, 43–51.
Brune, B., 2003. Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ. 10,
864–869.
Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E., 1992. Evidence for
decreasing quality of semen during past 50 years. BMJ 305, 609–613.
Fig. 9. Impacts of glyphosate and Roundup on the mRNA levels of aromatase, estrogen a and b and androgens receptors. After 24 h exposure of Leydig cells to various non-
cytotoxic concentrations of Roundup or glyphosate in 6-wells plates, the cell pellets were recovered and total RNA extracted by the method of TriReagent?(Sigma–Aldrich).
All operations for each concentration were repeated three times on three different manipulations. The SEMs are indicated in the figure (Anova test p < 0.001⁄⁄⁄⁄; p < 0.005⁄⁄⁄;
p < 0.01 and⁄⁄p < 0.05⁄).
10
É. Clair et al./Toxicology in Vitro xxx (2012) xxx–xxx
Please cite this article in press as: Clair, É., et al. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and
testosterone decrease at lower levels. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2011.12.009

Page 11

Chan, P., Mahler, J., 1992. NTP technical report on the toxicity studies of Glyphosate
(CAS No. 1071-83-6) Administered in Dosed Feed to F344/N Rats and B6C3F1
Mice. Toxic Rep Ser 16, 1-D3.
Cox, C., 1998. Herbicide factsheet – glyphosate (Roundup). J. Pesticide Reform 18, 3–
15.
Cox, C., 2004. Herbicide factsheet – glyphosate. J. Pesticide Reform 24, 10–15.
Crouch, S.P., Kozlowski, R., Slater, K.J., Fletcher, J., 1993. The use of ATP
bioluminescence as a measure of cell proliferation and cytotoxicity. J.
Immunol. Methods 160, 81–88.
Csipo, I., Montel, A.H., Hobbs, J.A., Morse, P.A., Brahmi, Z., 1998. Effect of Fas+ and
Fas? target cells on the ability of NK cells to repeatedly fragment DNA and
trigger lysis via the Fas lytic pathway. Apoptosis 3, 105–114.
Dallegrave, E., Mantese, F.D., Coelho, R.S., Pereira, J.D., Dalsenter, P.R., Langeloh, A.,
2003. The teratogenic potential of the herbicide glyphosate-Roundup in Wistar
rats. Toxicol. Lett. 142, 45–52.
Dallegrave, E., Mantese, F.D., Oliveira, R.T., Andrade, A.J., Dalsenter, P.R., Langeloh, A.,
2007. Pre- and postnatal toxicity of the commercial glyphosate formulation in
Wistar rats. Arch. Toxicol. 81, 665–673.
Gasnier,C.,Benachour,N.,Clair,E.,Travert,C.,Langlois,F.,Laurant,C.,Decroix-Laporte,
C., Seralini, G.E., 2010. Dig1 protects against cell death provoked by glyphosate-
based herbicides in human liver cell lines. J. Occup. Med. Toxicol. 5, 29.
Gasnier, C., Dumont, C., Benachour, N., Clair, E., Chagnon, M.C., Seralini, G.E., 2009.
Glyphosate-based herbicides are toxic and endocrine disruptors in human cell
lines. Toxicology 262, 184–191.
Gasnier, C., Laurant, C., Decroix-Laporte, C., Mesnage, R., Clair, E., Travert, C., Seralini,
G.E., 2011. Defined plant extracts can protect human cells against combined
xenobiotic effects. J. Occup. Med. Toxicol. 6, 3.
Gehin, A., Guillaume, Y.C., Millet, J., Guyon, C., Nicod, L., 2005. Vitamins C and E
reverse effect of herbicide-induced toxicity on human epidermal cells HaCaT: a
biochemometric approach. Int. J. Pharm. 288, 219–226.
Gerschenson, L.E., Rotello, R.J., 1992. Apoptosis: a different type of cell death. FASEB
J. 6, 2450–2455.
IFEN, 2007. Report on pesticides in waters. Data 2005.
Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A.A., Natale, C.,
Santacroce, R., Di Corcia, M.G., Lucchese, A., Dini, L., Pani, P., Santacroce, S.,
Simone, S., Bucci, R., Farber, E., 2002. Cell death: apoptosis versus necrosis
(review). Int. J. Oncol. 21, 165–170.
Lefevre, A., Saez, J.M., Finaz, C., 1983. HCG responsiveness of purified Leydig cells
from immature and mature rats. Horm. Res. 17, 114–120.
Leist, M., Jaattela, M., 2001. Four deaths and a funeral: from caspases to alternative
mechanisms. Nat. Rev. Mol. Cell Biol. 2, 589–598.
Liu, J.J., Wang, W., Dicker, D.T., 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.
Manas, F., Peralta, L., Raviolo, J., Garcia Ovando, H., Weyers, A., Ugnia, L., Gonzalez
Cid, M., Larripa, I., Gorla, N., 2009. Genotoxicity of AMPA, the environmental
metabolite of glyphosate, assessed by the Comet assay and cytogenetic tests.
Ecotoxicol. Environ. Saf. 72, 834–837.
Melaine, N., Lienard, M.O., Dorval, I., Le Goascogne, C., Lejeune, H., Jegou, B., 2002.
Multidrug resistance genes and p-glycoprotein in the testis of the rat, mouse,
Guinea pig, and human. Biol. Reprod. 67, 1699–1707.
Mesnage, R., Clair, E., Séralini, G.-E., 2010. Roundup in genetically modified crops:
regulation and toxicity in mammals. Theorie in der Ökologie 16, 31–33.
Monosson, E., 2005. Chemical mixtures: considering the evolution of toxicology and
chemical assessment. Environ. Health Perspect. 113, 383–390.
Nagai, A.,1992. Studyon ethane
spermatogenic damage and protective drugs against this damage in the rat.
Nippon Hinyokika Gakkai Zasshi 83, 1322–1329.
dimethanesulphonate(EDS)-induced
Petre-Lazar, B., Livera, G., Moreno, S.G., Trautmann, E., Duquenne, C., Hanoux, V.,
Habert, R., Coffigny, H., 2007. The role of p63 in germ cell apoptosis in the
developing testis. J. Cell. Physiol. 210, 87–98.
Popov, S.G., Villasmil, R., Bernardi, J., Grene, E., Cardwell, J., Wu, A., Alibek, D., Bailey,
C., Alibek, K., 2002. Lethal toxin of Bacillus anthracis causes apoptosis of
macrophages. Biochem. Biophys. Res. Commun. 293, 349–355.
Richard, S., Moslemi, S., Sipahutar, H., Benachour, N., Seralini, G.E., 2005. Differential
effects of glyphosate and roundup on human placental cells and aromatase.
Environ. Health Perspect. 113, 716–720.
Riechers, D.E., Wax, L.M., Liebl, R.A., Bush, D.R., 1994. Surfactant-increased
glyphosate uptake into plasma membrane vesicles isolated from common
Lambsquarters leaves. Plant Physiol. 105, 1419–1425.
Riss, T.L., 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.
Sarda-Mantel, L., Couturier, O., Rouzet, F., Vrigneaud, J.M., Bleichner-Perez, S., Le
Guludec, D., Chérel, M., 2004. Imagerie scintigraphique de l’apoptose. Médecine
Nucléaire – Imagerie fonctionnelle et métabolique 28, 383–391.
Savitz, D.A., Arbuckle, T., Kaczor, D., Curtis, K.M., 1997. Male pesticide exposure and
pregnancy outcome. Am. J. Epidemiol. 146, 1025–1036.
Skakkebaek, N.E., Rajpert-De Meyts, E., Main, K.M., 2001. Testicular dysgenesis
syndrome:an increasinglycommon
environmental aspects. Hum. Reprod. 16, 972–978.
Sprankle, P., Meggitt, W.F., Penner, D., 1975. Adsorption, mobility, and microbial
degradation of glyphosate in the soil. Weed Sci. 23, 229–234.
Stocco, D.M., King, S., Clark, B.J., 1995. Differential effects of dimethylsulfoxide on
steroidogenesis in mouse MA-10 and rat R2C Leydig tumor cells. Endocrinology
136, 2993–2999.
Tichy, M., Borek-Dohalsky, V., Rucki, M., Reitmajer, J., Feltl, L., 2002. Risk assessment
of mixtures: possibility of prediction of interaction between chemicals. Int.
Arch. Occup. Environ. Health 75 (Suppl.), S133–S136.
Toppari, J., Virtanen, H.E., Main, K.M., Skakkebaek, N.E., 2010. Cryptorchidism and
hypospadias as a sign of testicular dysgenesis syndrome (TDS): environmental
connection. Birth Defects Res. A Clin. Mol. Teratol. 88, 910–919.
Travert, C., Carreau, S., 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.
Tribull, T.E., Bruner, R.H., Bain, L.J., 2003. The multidrug resistance-associated
protein 1 transports methoxychlor and protects the seminiferous epithelium
from injury. Toxicol. Lett. 142, 61–70.
U.S. EPA, 1998. Environmental Protection Agency. Endocrine Disruptor Screening
and Testing Advisory Committee (ECSTAC) Final Report. Washington, DC.
Uezono, T., Maruyama, W., Matsubara, K., Naoi, M., Shimizu, K., Saito, O., Ogawa, K.,
Mizukami, H., Hayase, N., Shiono, H., 2001. Norharman, an indoleamine-derived
beta-carboline, but not Trp-P-2, a gamma-carboline, induces apoptotic
cell death in human neuroblastoma SH-SY5Y cells. J. Neural Transm. 108,
943–953.
Vecchio, L., Cisterna, B., Malatesta, M., Martin, T.E., Biggiogera, M., 2004.
Ultrastructural analysis of testes from mice fed on genetically modified
soybean. Eur. J. Histochem. 48, 448–454.
Vilches Troya, J., 2005. Understanding cell death: a challenge for biomedicine. An. R
Acad. Nac. Med. (Madr.) 122, 631–656, discussion 656–639.
Walsh, L.P., McCormick, C., Martin, C., Stocco, D.M., 2000. Roundup inhibits
steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein
expression. Environ. Health Perspect. 108, 769–776.
Yousef, M.I., Salem, M.H., Ibrahim, H.Z., Helmi, S., Seehy, M.A., Bertheussen, K., 1995.
Toxic effects of carbofuran and glyphosate on semen characteristics in rabbits. J.
Environ. Sci. Health B 30, 513–534.
developmentaldisorder with
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Please cite this article in press as: Clair, É., et al. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and
testosterone decrease at lower levels. Toxicol. in Vitro (2012), doi:10.1016/j.tiv.2011.12.009