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Integrative Pharmacology, Toxicology and Genotoxicology

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The toxicity of the active molecule in herbicides has been used to determine safe concentrations, because other components are considered inert. Roundup, which contains the active molecule Glyphosate, was described as an endocrine disrupter because non-cytotoxic concentrations inhibited progesterone synthesis in vitro. Human chorioplacental JAr cells synthesise progesterone, and increase synthesis when stimulated by chorionic gonadotrophin (hCG), or the transduction molecule cAMP. JAr cells were exposed to two Roundup formulations, and compared with the same concentrations of glyphosate ± cAMP, or ± hCG for 1, 4, 24, 48 or 72h. The surviving viable cells were quantified using an MTT assay, and progesterone was measured in an ELISA. hCG and cAMP stimulated progesterone synthesis by cells in vitro as expected. In contrast to previous reports, JAr cell death preceded decreased progesterone synthesis, and steroidogenesis was unaffected by low, non-cytotoxic concentrations of Roundup or glyphosate. Roundup was more cytotoxic than glyphosate alone; the 24h EC50 was 16mM for glyphosate, but 0.008mM when glyphosate was in a 7.2g/L Roundup formulation. Significant cytotoxicity was caused by glyphosate in Roundup (p<0.01) after 24h, and cytotoxicity was observed in vitro after exposure toa range of concentrations comparable to the Australian Drinking Water Guidelines. Endocrine disruption effects were secondary to cytotoxicity. Roundup was more cytotoxic than the same concentration of glyphosate alone, indicating that the other constituents of the herbicide are not inert. There is a compelling need to conduct in vivo studies to characterise the toxicity of glyphosate in a Roundup formulation, to facilitate re-evaluation of existing public health guidelines.
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Research Article
Integrative Pharmacology, Toxicology and Genotoxicology
Integr Pharm Toxicol Gentocicol, 2015 doi: 10.15761/IPTG.1000104 Volume 1(1): 12-19
Endocrine disruption and cytotoxicity of glyphosate and
roundup in human JAr cells in vitro
Fiona Young*, Dao Ho, Danielle Glynn and Vicki Edwards
Department of Medical Biotechnology, Flinders University, Adelaide, South Australia
Abstract
e toxicity of the active molecule in herbicides has been used to determine safe concentrations, because other components are considered inert. Roundup, which
contains the active molecule Glyphosate, was described as an endocrine disrupter because non-cytotoxic concentrations inhibited progesterone synthesis in vitro.
Human chorioplacental JAr cells synthesise progesterone, and increase synthesis when stimulated by chorionic gonadotrophin (hCG), or the transduction molecule
cAMP.
JAr cells were exposed to two Roundup formulations, and compared with the same concentrations of glyphosate ± cAMP, or ± hCG for 1, 4, 24, 48 or 72h. e
surviving viable cells were quantied using an MTT assay, and progesterone was measured in an ELISA.
hCG and cAMP stimulated progesterone synthesis by cells in vitro as expected. In contrast to previous reports, JAr cell death preceded decreased progesterone
synthesis, and steroidogenesis was unaected by low, non-cytotoxic concentrations of Roundup or glyphosate. Roundup was more cytotoxic than glyphosate alone;
the 24h EC50 was 16mM for glyphosate, but 0.008mM when glyphosate was in a 7.2g/L Roundup formulation. Signicant cytotoxicity was caused by glyphosate
in Roundup (p<0.01) after 24h, and cytotoxicity was observed in vitro after exposure toa range of concentrations comparable to the Australian Drinking Water
Guidelines.
Endocrine disruption eects were secondary to cytotoxicity. Roundup was more cytotoxic than the same concentration of glyphosate alone, indicating that the other
constituents of the herbicide are not inert. ere is a compelling need to conduct in vivo studies to characterise the toxicity of glyphosate in a Roundup formulation,
to facilitate re-evaluation of existing public health guidelines.
Correspondence to: Dr. Fiona Young, Department of Medical Biotechnology,
Flinders University, Flinders Drive, Bedford Park, Adelaide, South Australia, Tel:
+61 8 7221 8558, E-mail: Fiona.Young@inders.edu.au
Key words: Glyphosate N-phosphonomethyl glycine, roundup, endocrine disrupting
compound, Progesterone, Steroidogenic Acute Regulatory Protein (StAR), Human
Chorionic Gonadotrophin (HCG)
Received: January 04, 2015; Accepted: February 02, 2015; Published: February
10, 2015
Introduction
Endocrine disrupting compounds (EDCs) are dened by the U.S.
Environmental Protection Agency (EPA) as being “exogenous agents
that interfere with synthesis, secretion, transport, metabolism, binding
action, or elimination of natural blood-borne hormones that are present
in the body.” e US Endocrine Society noted that key mechanisms of
action for endocrine disruptioninvolve perturbation of the enzymatic
pathways involved in steroid biosynthesis [1].
e rate limiting step of steroid hormone synthesis is the uptake
of cholesterol from the outer to the inner mitochondrial membrane, a
process mediated by the steroidogenic acute regulatory protein (StAR)
[2,3]. Once cholesterol is inside the mitochondria it is rapidly converted
to pregnenolone by cytochrome P450 cholesterol side chain cleavage
enzyme (P450scc), and pregnenoloneis converted to progesterone by
3β-hydroxysteroid dehydrogenase (3β-HSD) [4,5]. e StAR protein
is acutely regulated, dependent on trophic hormone stimulation
and is not an enzyme, which makes it more of a target for endocrine
disruption than the steroidogenic enzymes, which are usually present
in excess amounts and have long half-lives [2]. e cytochrome P450
aromatase enzyme (P450arom), which produces estradiol 17 beta (E2),
also appears to be modulated by xenobiotics such as Roundup[6,7].
Both StAR and P450arom are upregulated by activation of the
luteinising hormone/chorionic gonadotrophin receptor (LH /CG),
which has cyclic adenosine monophosphate (cAMP) as an intracellular
transduction molecule. e administration of a cAMP analogue,
dibutrylcAMP, to the steroidogenic MA10 cell line in vitro upregulated
StAR protein and stimulated a four-fold increase in synthesis of the
steroid hormone progesterone [8].
e human JAr cell line was derived from a choriocarcinoma
tumour of the placenta [9] and maintains placental and endocrine
functions in vitro [10,11] including the synthesis and secretion of
progesterone (P4), 17β-estradiol and human chorionic gonadotrophin
(hCG), as well as the transport of nutrients and waste products in
and out of the cell: functions which are characteristic of the placental
transport from mother to foetus [10,12,13]. e hepatobiliary-
like characteristics of the JAr cell line make them a good model for
studying the uptake, activation and elimination of novel compounds
in vitro [11,14]. JAr cells also proliferate rapidly in vitro, with reported
doubling times ranging from 15h [15] to 29h [16]. e steroidogenicJAr
cell line therefore can be used to investigateEDCs that disrupt hormone
synthesis [14,17].
Glyphosate inhibits the 5-enolpyruvoylshikimate-3-phosphate
Young F (2015) Endocrine disruption and cytotoxicity of glyphosate and roundup in human JAr cells in vitro
Volume 1(1): 12-19Integr Pharm Toxicol Gentocicol, 2015 doi: 10.15761/IPTG.1000104
synthase enzyme found in plant, but not animal aromatic amino acid
synthesis pathways. is specicity to plants, along with its metabolic
breakdown to AMPA and thereaer to CO2, provided rationale for
its development as a herbicide. e Monsanto company utilised
these properties by developing transgenic maize, cotton and other
crops that are resistant to glyphosate [18], thus increasing broad scale
application of glyphosate in Roundup formulations.Glyphosate occurs
as a hydrophilic acidic isopropylamine salt [19], which impairs its entry
to lipid membrane bound cells. e herbicidal activity of glyphosate
is increased by adding surfactants and other adjuvants, and a number
of these mixtures are marketed under the blanket term ‘Roundup’
[20,21]. ere are a number of Roundup formulations containing
dierent concentrations of glyphosate and adjuvants such as the
surfactant POEA [21]. e adjuvants and ‘inert’ constituents of many
of these formulations have been identied, and the dierent Roundup
formulations assigned to dierent classes of high, mid and low toxicity
accordingly [22]. Roundup Bioforce with 360g/L glyphosate, and
another formulation with 7.2g/L glyphosate, are amongst the least toxic
Roundup formulations.
Manufacturers recommend that 1-2% solutions of Roundup in
water should be used for agricultural applications, hence agricultural
workers may be exposed to 100% concentrated solutions of
approximately 2.13M glyphosate, and to 1-2% dilutions of the 360g/L
Roundup formulation, which equates to exposure to 21-42mM of
glyphosate. Roundup for domestic applications is sold in ready-to-
use sprays, or in concentrated forms that require dilution to similar
concentrations to those used in the agricultural setting. e Australian
and New Zealand Environment and Conservation Council [23] gives
0.2mg/L (0.001mM) glyphosate as the trigger value for recreational
water quality (2000), and the Australian Drinking Water Guideline
(NH&MRC 2011) [24] is 1mg/L (0.0059mM) because this is 10% of
the acceptable daily intake and does not pose a risk to human health.
e USEPA and other jurisdictions regulate the concentration
of the active ingredient glyphosate, but the adjuvants in Roundup
are considered to be inert and hence are not required to be subjected
to toxicological assessment [21,22,25]. Recent reports however,
indicate that the toxicity of Roundup is not directly correlated to
the concentration of glyphosate, but to the diering adjuvants and
surfactants comprising various Roundup formulations [7,20].
Serum-free in vitro exposure conditions were used to determine
that a 2h exposure to 0.15mM glyphosate in a 180g/L Roundup
formulation did not aect mouse Leydig MA10 cell viability but did
signicantly reduce progesterone dose-dependently at non-cytotoxic
concentrations [26]. Although total protein synthesis was not aected,
StAR protein expression was reduced by 90%, CYP450scc activity
was halved, and progesterone synthesis was halved by the Roundup,
whereas glyphosate had no eect at 100µg/mL (0.6mM), the highest
dose tested [26].
Similarly, a short in vitro serum-free exposure to 0.015mM
glyphosate in a 360g/L Roundup formulation (i.e. dierent adjuvents
from the 180g/L formulation) halved the viability of human placental
choriocarcinoma JEG3 cells [6]. Although the diering toxicity of the
two Roundup formulations [20], and the diering sensitivity of the
two cell lines (Leydig MA10 and chorioplacental JEG3) to xenobiotics
prevent direct comparisons between these two studies, it can be
concluded that Roundup, but not Glyphosate, reduced progesterone
synthesis by inhibiting steroidogenic enzyme expression and activity,
and that these endocrine disrupting eects occurred in the absence of
cell death. Furthermore, endocrine disruption in these in vitro models
occurred aer relatively short 2-18h exposures and at concentrations in
the same range that agricultural workers are exposed to when applying
1-2% solutions of Roundup.
e addition of serum to in vitro cell culture systems reduced the
toxicity of Roundup [6]. e cell culture media were acidied to pH 5.8
for these experiments [19]. e P450arom activity was related to pH: as
acidity increased (pH decreased), enzyme activity decreased.
In serum-free pH 5.8 in vitro culture conditions, the cytotoxic
mechanism of action of Roundup and glyphosate wasapoptosis
followed by secondary necrosis. Roundup caused general cell plasma
membrane damage before decreasing mitochondrial membrane
succinate dehydrogenase activity as measured in the MTT assay.
In contrast, glyphosate did not initially reduce plasma membrane
integrity, and only decreased succinate dehydrogenase activity[20].
e mixtures of glyphosate and adjuvants in Roundup have not
been examined in vivo, except in one well-conducted 2 year chronic
toxicity study [27,28], in which serum concentrations of estrogen
and androgen in rats exposed to low concentrations of Roundup in
drinking water were reported. Roundup aected the ratio of these two
hormones in vivo, and there was an increase in estrogen-dependent
mammary tumour formation [29].
ere is only one report describing the eect of glyphosate and
Roundup on progesterone synthesis [26], and this study used a cell
line derived from a male mouse. Progesterone is essential for the
correct regulation of the human menstrual cycle and for maintaining
pregnancy, and the eect of the ubiquitous herbicide Glyphosate, or
Roundup, on progesterone synthesis by human female cells requires
further investigation. Progesterone synthesis in the second half
of the menstrual cycle in vivo is regulated by LH, and high levels of
progesterone synthesis are maintained during pregnancy in vivo by
embryonic secretion of human chorionic gonadotrophin (hCG), which
binds to the common transmembrane LH/hCG receptor. e eects
of Roundup on progesterone secretion by human female cells, and the
activity of the pituitary-derived regulatory gonadotrophin, luteinising
hormone (LH), and embryo-derived chorionic gonadotrophin (CG)
activity, have not previously been examined in vitro or in vivo.
Walsh et al. (2000) [26] concluded that the inhibitory eect of
Roundup on progesterone production by the male mouse Leydig
MA10 cell line was mediated by downregulation of the steroidogenesis
rate-limiting protein, StAR. Our study aims to conrm this observation
by exposing the human female chorioplacental JAr cell line (similar to
the previously examined JEG cell line) to glyphosate and Roundup in
vitro in order to measure cell viability and eects on steroidogenesis.
Secondly, we aim to use the LH/hCG receptor transduction molecule
cAMP to upregulate progesterone synthesis. Since LH/hCG receptor
binding upregulates intracellular cAMP, which in turn upregulates
StAR [30,31] which stimulates progesterone synthesis by JAr cells in
vitro [14], progesterone can be used to indirectly monitor StAR activity.
We hypothesise that non-cytotoxic concentrations of Roundupwill
inhibit basal progesterone synthesis at lower concentrations than
Glyphosate, and that non-toxic concentrations of Roundup, but not
Glyphosate, will attenuate cAMP-stimulated progesterone synthesis.
is hypothesis is supported by Clair et al. [32], who found that
Roundup decreased testosterone production by primary derived
testicular cells at non-cytotoxic concentrations.
is will be the rst study to examine the eects of glyphosate and
Young F (2015) Endocrine disruption and cytotoxicity of glyphosate and roundup in human JAr cells in vitro
Volume 1(1): 12-19Integr Pharm Toxicol Gentocicol, 2015 doi: 10.15761/IPTG.1000104
Roundup on progesterone production by human female cells in an in
vitro cell culture system that models key aspects of reproduction in
women.
Materials and methods
Chemicals
All chemicals and reagents used in this study were HPLC grade and
obtained from Sigma-Aldrich unless otherwise stated.
Cell line and cell culture
e JAr cell line [9] was obtained from the Global Bioresource
CentreTM (ATCC) and maintained in RPMI-1640 medium
supplemented with 10% heat inactivated Foetal Calf Serum (FCS,
Invitrogen Corporation), sodium pyruvate (1mM), HEPES (10mM),
glucose (4.5g/L), L-glutamine (2mM), sodium bicarbonate (1.5g/L),
penicillin (60mg/L) and streptomycin (50mg/L), at 37°C in humidied
atmosphere with 5% CO2 andsubcultured every 2-3 days as required.
For all experiments, exponentially growing cells (80% conuence) were
detached from asks with 0.25% Trypsin-EDTA solution. Cell number
and viability was determined using the trypan blue exclusion assay on
a haemocytometer before each experiment [33].
Glyphosate and roundup and JAr cell exposure
Glyphosate N-phosphonomethyl glycine was initially dissolved
in water then diluted with RPMI+10% FCS such that the nal
concentration of RPMI was 97% v/v.Two Roundup formulations
(7.2g/L ‘Regular Roundup Weedkiller’ and 360g/L glyphosate ‘Roundup
Concentrate Weedkiller’) were examined in this study, both obtained
over-the-counter fromWoolworths Pty Ltd, Australia. Information
about the ‘inert’ ingredients was not disclosed by the manufacturers.
A concentrated stock solution of each Roundup formulation was
produced, such that the nal concentration of RPMI was 97% v/v.
RPMI medium with 10% FCS was diluted to 97% v/v with RO water
to serve as a ‘vehicle’ control. All media, glyphosate and Roundup
solutions were adjusted to pH 7.4.
In the rst experiment, seven 1:10 serial dilutions of 7.2g/L
glyphosate in RPMI+10%FCS, or the same concentration of glyphosate
in a 7.2g/L (45.6mM) ‘Regular Roundup Weedkiller’ Roundup
formulation, with or without dibutryl cyclic adenosine monophosphate
(cAMP, 1mM, activates the LH/hCG transduction pathway), were
prepared. e JAr cells were pre-cultured for 2h in 100% RPMI+FCS
to facilitate adherence to the culture vessel, these media were discarded,
and the cells were exposed to each concentration of glyphosate or
Roundup ± cAMP in triplicate wells for 24 or 72h before media
were collected for progesterone (P4) measurement by ELISA, and the
numbers of surviving viable cells were determined by MTT assay. is
experiment was repeated on three separate occasions (n=3).
To further explore the cytotoxicity of Roundup in the presence of
hCG, JAr cells (20,000 cells per well) were pre-cultured for 2h before
exposure to the same seven 1:10 serial dilutions of 7.2g/L glyphosate
in ‘Regular Roundup Weedkiller’ Roundup formulation ± hCG
(1000mIU/mL) for 1, 4, 24 or 48h. Each concentration or control
treatment was examined in triplicate wells on three separate occasions
(n=3). Media were discarded and the numbers of viable cells remaining
in the wells determined by MTT assay.
In the third experiment,a Roundup Weed Killer Concentrate
with 360g/L (2.13M) Glyphosate N-phosphonomethyl glycine + 10%
surfactant, or Glyphosate N-phosphonomethyl glycine, were diluted
with RPMI+10%FCS to 0.05M, such that the nal concentration of
RPMI was 97% v/v. Each 0.05M stock solution was diluted in RPMI+10%
FCS medium to generate solutions of 5x10-6M, 5x10-5M, 1x10-4M,
2.5x10-4M, 5x10-4M, 1x10-2M. e JAr cells were preincubated for 2h
before exposure to glyphosate, or the same concentration of glyphosate
in Roundup concentrate formulation, for 24h. Each concentration or
control treatment was examined in triplicate wells on three separate
occasions (n=3). e media were collected for progesterone (P4)
measurement by ELISA, and the numbers of surviving viable cells were
determined by MTT assay.
MTT cytotoxicity assay
iazolyl blue tetrazolium bromide (MTT) was dissolved in
sterile PBS to give a nal concentration of 5mg/ml. e MTT assay
was carried out by modication of the original Mosmann (1983) assay
[34,35]. Standard curves were generated for each replicate experiment,
which consisted of 6 serial dilutions spanning 0-100,000 cells per well
in 96 well plates. Each of the 6 cell concentrations was examined in 6
replicate wells. JAr cells in the standard curve plates were incubated
for 24h before media were discarded and the number of viable cells per
well determined.
For the MTT assay the stock MTT was diluted in RPMI+10%FCS
to 0.5mg/ml. Culture and treatment media were discarded and 100µl
MTT were added to each treatment or standard curve well for 1h at
37°C + 5% CO2. Aer this, 80µl of 20% SDS in 0.02M HCl were added
to each well for 1h at room temperature. e absorbance was measured
at 570nm, with reference absorbance 630nm, using an automatic
spectrophotometer with KC Junior soware.
Progesterone enzyme linked immunosorbent assay (ELISA)
e primary antibody against progesterone (P4) was used to coat
the wells of a Maxisorb 96-well ELISA plate (Nunc). ‘Blank’ ELISA
control wells contained 50µl ELISA Immuno Assay (EIA) buer but
no primary antibody, whereas the reference standard wells contained
25µl puried P4(0-4 ng/ml) prepared in RPMI 1640 medium, and
the test wells contained 25µl conditioned cell culture medium. 25µl
Glyphosate and Roundup solutions in RPMI+10%FCS (5x10-6M, 5x10-
5M, 1x10-4M, 2.5x10-4M, 5x10-4M, 1x10-2M) were also added to single
test wells to determine if the herbicides interfered with the ELISA. e
standard and test wells had 25µl EIA buer added, then all the wells
had 50µl horseradish peroxidase (HRP) conjugated to P4 added before
incubation at room temperature for 100 minutes. Aer washing, 100µl
of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS),
hydrogen peroxide and substrate buer were added to each well for
one hour or until the highest standards reached an absorbance of 1.0.
Absorbance was read at primary wavelength 405nm, and reference
wavelength 540nm using KC Junior soware. In the zero standard
wells the HRP-conjugate bound directly to the primary antibody, and
subsequently generated the highest optical density (OD) value. In the
wells containing the highest concentration of reference standard, the
standard competed with the HRP-conjugate for antibody binding sites,
and hence generated the lowest OD values. Hormone concentration
in test wells was calculated by comparison with the standard curve.
Coecients of variation for this ELISA were described previously [14].
Statistical analysis
e numbers of viable cells per well aer 24h incubation in 97%
and 100% RPMI+10% FCS were compared using a 2-tailed unpaired
Students T-test with signicance assigned at p<0.05. e cytotoxicity
Young F (2015) Endocrine disruption and cytotoxicity of glyphosate and roundup in human JAr cells in vitro
Volume 1(1): 12-19Integr Pharm Toxicol Gentocicol, 2015 doi: 10.15761/IPTG.1000104
and the hormone values were subjected to 1-way ANOVA and Tukey
post-hoc test, or to 2-way ANOVA with Bonferroni post-hoc test, with
signicance assigned at p<0.05. e EC50 for cytotoxicity and hormone
synthesis, were determined using GraphPad Prism.
Results
e numbers of viable JAr cells in the 97% RPMI+10% control
medium were 4810 ± 775 cells per well (mean ± stdev) aer 24h in vitro
culture (Figure 1), and 33364 ± 6249 cells per well aer 72h culture. e
addition of cAMP had no signicant eect on JAr cell proliferation aer
24h (4787 ± 1432) or 72h (21025 ± 3309). e highest concentration of
glyphosate tested (720mg/L) did not aect JAr cell viability whereas
the highest concentration of Roundup tested (containing 720mg/L
glyphosate) killed all the JAr cells aer 24 and 72h (p<0.001, Figure 1).
is formulation of Roundup caused signicant cytotoxicity aer a 24h
exposure to 0.72mg/L (p<0.01). e cell viability EC50 for exposure to
the 7.2g/L formulation of Roundup was 1.3mg/L and 0.9mg/L aer 24h
for basal and cAMP-stimulated cells respectively, and 0.29mg/L aer
72h for both basal and cAMP-stimulated cells.
Basal progesterone production by JAr cells cultured in 97%RPMI
and 10%FCS was 1.46 ± 0.2 ng/mL aer 24h, and 8.32 ± 1.5ng/mL
aer 72h (Figure 2). Cyclic AMP signicantly stimulated progesterone
production to 1.97 ± 0.3 (p<0.01) and 14.89 ± 3ng/mL (p<0.001) aer
24 and 72h respectively. Glyphosate did not aect basal or cAMP
stimulated progesterone synthesis aer 24 or 72h exposure, but
0.072mg/L Roundup signicantly reduced basal (1.18 ± 0.13) and
cAMP-stimulated (1.74 ± 0.14) progesterone synthesis (p<0.05) aer
24h exposure. Exposure to 0.72mg/L Roundup for 72h had no eect
on basal progesterone synthesis, but signicantly reduced cAMP-
stimulated progesterone synthesis to 12 ± 1.6ng/mL (p<0.01). e IC50
values for basal and cAMP-stimulated progesterone synthesis aer 24h
exposure to Roundup were 0.2 and 0.8mg/L respectively, and 1.1mg/L
and 1.4mg/L for basal and cAMP stimulated progesterone production
respectivelyaer 72h exposure.
e cytotoxicity caused by Roundup was not aected by hCG (2-
way ANOVA, Figure 3). A 1h exposure to 72mg/L (0.42mM) glyphosate
in Roundup formulation caused signicant cytotoxicity, and exposure
to 0.042mM caused signicant cytotoxicity aer 4h (Figure 3). e
EC50 value aer a 24h exposure to Roundup and hCG was 0.007mM,
similar to the 0.005mM EC50 value aer 24h exposure to Roundup and
cAMP (Table 1).
In a second series of experiments using a Roundup formulation
with 320g/L glyphosate, there was no dierence in JAr cell viability
when cultured in 100% or 97% RPMI media (Figure 3), and the JAr
cell numbers increased from 40000 to 71946 ± 8792 cells per well (n=7)
in 24h. Roundup caused signicant cytotoxicity at 0.1mM (p<0.05),
whereas glyphosate caused cytotoxicity at a concentration that was two
orders of magnitude higher (10mM, p<0.001). e cytotoxicity IC50
values were 16 and 0.13mM for glyphosate and Roundup respectively.
Progesterone synthesis was signicantly decreased by 2.5mM Roundup
(p<0.01) and 50mM Glyphosate (p<0.01). Cell death was caused
by lower concentrations of Glyphosate or Roundup than those that
inhibited progesterone production (Figure 3).
Cells per Well
Glyphosate (mg/L)
Figure 1. Effect of Glyphosate or Roundup (7.2g/L) on JAr cell viability.
Human JAr cells were exposed to glyphosate ( ) or Roundup (, formulation contained
7.2g/L glyphosate) ± cAMP (----+----) in triplicate wells in 96 well plates on three separate
occasions (n=3) for 24h or 72h. The mean±stdev numbers of surviving cells in each well
were determined in an MTT assay, by comparison with a standard curve generated for each
experimental replicate. Data analysed by 2-way ANOVA with Tukey post-hoc test, and
difference from controls shown; Roundup p<0.01 **, p<0.001 ***.
Progesterone per well (ng/mL)
Glyphosate (mg/L)
Figure 2. Effect of Glyphosate or Roundup (7.2g/L) on JAr cell Progesterone synthesis.
Human JAr cells were exposed to glyphosate ( ) or Roundup (, formulation contained
7.2g/L glyphosate) in triplicate wells in 96 well plates on three separate occasions (n=3) for
24h or 72h with (- -+- -) or without cAMP. The progesterone concentration (ng/mL) in each
well was determined in an ELISA assay, by comparison with a standard curve, and the mean
± stdev concentration of three independent experimental replicates (n=3) shown. Data
analysed by 2-way ANOVA with Bonferroni post-hoc test to compare cAMP-stimulated or
basal progesterone secretion against glyphosate dose, and difference from control shown;
p<0.05*, p<0.01 **, p<0.001 ***.
Young F (2015) Endocrine disruption and cytotoxicity of glyphosate and roundup in human JAr cells in vitro
Volume 1(1): 12-19Integr Pharm Toxicol Gentocicol, 2015 doi: 10.15761/IPTG.1000104
Discussion
Human reproductive cells were exposed to two dierent
formulations of Roundup, and their eects on basal, cAMP- and
hCG-stimulated cell viability and progesterone production were
examined for the rst time. e in vitro model included serum and was
conducted at physiological pH. Transformed cell lines are less sensitive
than primary-derived cells in vitro [20,21], and therefore provide
conservative estimates for potential cytotoxicity in vivo.
ese in vitro cell culture systems did not model in vivo absorption,
distribution, metabolic or excretory parameters, northe regulation of
serum carrier and binding proteins. e concentration of FCS (10%)
used in the present study was too low to model the 100% equivalent
concentration of serum components in vivo, but was used to allow
comparison with other cell culture studies. e inclusion of 10% serum
in vitro delayed the toxic eects of Roundup by approximately 24h
previously [6], and in the present study (Table 1). In acute exposures
therefore, increasing serum concentrations in vitro decreases the toxic
eects of Roundup.
e data from our study conrmed previous observations [22]
that glyphosate caused cytotoxicity at concentrations at least two
orders of magnitude higher than the equivalent concentration of
glyphosate in Roundup (Table 1). e cytotoxicity EC50 values for
a range of cell lines exposed to glyphosate for 18-24h in vitro ranged
from 16 to 37mM, whereas exposure to the same concentration of
glyphosate in a Roundup formulation (with 7.2g/L glyphosate in the
parent solution) generated EC50 values ranging from 0.002 to 0.38mM
(Table 1). e EC50 values for cells exposed to Roundup with 360g/L
in the parent solution for 24h ranged from 0.13 to 27.6mM (Table
1). e EC50 values for Roundup spanned two orders of magnitude,
whereas the dierences in cell lines and culture conditions had less
eect on the Glyphosate EC50 values; observations partially explained
by the membrane-disrupting mechanism of action of Roundup. e
data in Table 1 support the proposal that when serum is present in
vitro, increasing pH from acidic to neutral physiological levels further
decreases Roundup cytotoxicity.
In initial experiments the presence of cAMP appeared to increase
JAr cell sensitivity to Roundup when measured by both MTT assay and
progesterone synthesis (Table 1) aer a 24h, but not a 72h exposure.
When this was examined in more detail by exposing cells to Roundup
and hCG for shorter exposure periods however, it was found that
hCG did not aect the cytotoxicity of Roundup. e EC50 values
for cytotoxicity were similar for hCG and its secondary messenger
molecule cAMP, as might be expected.
e LH/hCG transduction molecule cAMP also stimulated
progesterone synthesis as expected. Walsh et al. [8] demonstrated that
cAMP upregulated the StAR protein whichincreased progesterone
production, and that low non-cytotoxic concentrations of Roundup
inhibited cAMP-stimulated StAR upregulation, and caused associated
decreases in progesterone production. In our study however, the
inhibition of cAMP-stimulated progesterone synthesis occurred at
higher, cytotoxic concentrations of Roundup. ese data suggest that
the two formulations of Roundup that we tested did not act directly
on the StAR protein when they were tested at physiological pH and
in the presence of serum, but that the reductions in both basal and
cAMP-stimulated progesterone synthesis were caused by the loss of
viable steroidogenic cells. In our study, the inhibition of progesterone
secretion did not precede cytotoxicity, and endocrine disruption eects
were a consequence of cell death. Others have demonstrated that
Roundup downregulated P450arom expression and activity [7] in low
non-physiological pH and serum-free in vitro culture conditions, but
the synthesis of estradiol at physiological pH and in the presence of
serum has not yet been examined. Given this lack of data, the proposal
that Roundup has endocrine disrupting activity independent of its
cytotoxic activity, needs further study.
Previously, a short two hour exposure to Roundup generated a
higher EC50 value (0.14mM) for progesterone synthesis [8] than our
longer 24h exposure (0.012mM). e presence of serum in our culture
system probably reduced the toxicity of Roundup and in this way caused
the lower EC50 value. In our study, the EC50 value for progesterone
Cells per Well
Glyphosate (mg/L)
Figure 3. Effect of Roundup (7.2g/L) and hCG on JAr cell viability.
Human JAr cells were exposed to Roundup (formulation contained 7.2g/L glyphosate) with
(■) or without () human chorionic gonadotrophin (hCG, 1000mIU/mL) in triplicate wells
in 96 well plates on three separate occasions (n=3) for 1, 4, 24 or 48h. The mean±stdev
numbers of surviving cells in each well were determined in an MTT assay, by comparison
with a standard curve generated for each experimental replicate. Data analysed by 2-way
ANOVA with Bonferroni post-hoc test, to compare hCG-stimulated or basal cell viability
against glyphosate dose, and difference from control shown; p<0.01**, p<0.001***.
Young F (2015) Endocrine disruption and cytotoxicity of glyphosate and roundup in human JAr cells in vitro
Volume 1(1): 12-19Integr Pharm Toxicol Gentocicol, 2015 doi: 10.15761/IPTG.1000104
synthesis was higher than for cytotoxicity (Table 1), indicating that the
signicant decreases in progesterone concentrations were caused by
reduced numbers of viable cells.
e two Roundup formulations selected for examination in this
study are available for purchase from local supermarkets for use in
domestic gardens. Members of the public may be exposed to these
formulations of Roundup by aerosol or dermal contact, for perhaps 1
– 2 hours, at relatively low frequencies of perhaps 2 to 8 times per year,
and the eect of these exposures on healthy adults who use Roundup
domestically are not known. It is possible that children, pregnant
women, the elderly and those suering chronic illnesses, may be
more vulnerable to environmental toxic insults such as that caused by
exposure to Roundup sprays than the general population. Irrespective
of public exposures, the Australian Drinking Water Guideline (2011)
for glyphosate is 0.0059mM, and is based on the premise that an adult
of average weight who drinks 2L of water a day would not be harmed
if the water contained 0.0059mM glyphosate(NH&MRC 2011). e
ADWG (2011) notes that ‘excursions above this level would need to
occur over a signicant period to be of health concern, as the health-
based guideline is based on long-term eects’(NH&MRC 2011). e
in vitro EC50 values for glyphosate alone are two or three orders of
magnitude higher than this Guideline level (Table 1), and originally
justied the use of this concentration, but in our study the 24h basal
in vitro EC50 value for glyphosate in Roundup formulation was
0.008mM, and in the presence of the cAMP was 0.005mM. Cell lines
are less sensitive than primary derived cells such as the epithelial cells
lining the gastrointestinal (GI) tract, and low pH conditions such as
those found in the upper GI tract increase Roundup cytotoxicity. Both
of these factors may therefore increase the toxicity of Roundup in vivo.
Conversely, Glyphosate has low absorption from the GI tract (30-36%)
in rats and rabbits (Council 2011), and the higher proportion of serum
constituents in vivo may reduce Roundup toxicity. Seralini et al. [28]
demonstrated that 50ng/L Roundup in drinking water had toxic eects
in a 2y in vivo study, which constitutes a study of long term eects,
but at concentrations much lower than the 1mg/L Australian Drinking
Water Guideline.
In Australia and other countries, drinking water is commonly
produced by chlorination of supplies from protected catchments,
and/or by Dissolved Air Flocculation and Filtration followed by
chlorination, but it is not clear how eciently these process remove the
adjuvants found in Roundup, nor if the specic surfactants and other
adjuvants comprising the dierent formulations of Roundup persist
into the public water supply. It is also not known whether the chronic
toxic eects seen in vivo [28] were caused by the supposedly inert
surfactant components of Roundup, or the combination of Roundup
surfactants with glyphosate. In the absence of robust data it would be
prudent to consider the possibility that Roundup surfactants may be
found in drinking water that also contains glyphosate, and to evaluate
these mixtures in vivo, in order to assess the continuing relevance of
the 1mg/L Glyphosate Drinking Water Guideline level. ere isclearly
an urgent need to conduct in vivo studies to determine the acute and
chronic toxicity of glyphosate in domestic Roundup formulations, in
order to ensure that existing drinking water guidelines are safe.
Conclusions
Glyphosate alone is less toxic than glyphosate in a Roundup
formulation; both glyphosate and Roundup caused cell death
which resulted in decreased progesterone levels in vitro, and
Cell Line Culture conditions, exposure, pH
& FCS
Concentration of Glyphosate
in the Roundup Formulation
EC50 Glyphosate (mM) EC50 same concentration of
Glyphosate in Roundup (mM)
Reference
Cytotoxicity measured in an MTT mitochondrial succinate dehydrogenase assay.
JEG3 Serum-free, pH 5.8, 18h 360g/L 25.2 4.2 Richard, et al. [6]
JEG3 10% FCS, pH 5.8, 18h 360g/L 27.3 16.8 Richard, et al. [6]
JEG3 10% FCS, pH 5.8, 24h 360g/L 37.2 27.6 Benechour, et al. [19]
HEK293 10% FCS, pH 5.8, 24h 360g/L 34 17 Benechour ,et al. [19]
JEG3 Serum-free, pH na, 24h 360g/L ~7.56 na Mesnage, et al. [21]
JEG3 Serum-free, pH 5.8, 24h 7.2g/L ~42.6 ~0.38 Benechour, et al. [20]
JEG3 Serum-free, pH 5.8, 24h 360g/L ~42.6 ~6.39 Benechour, et al. [20]
JAr 10% FCS, pH 7.4, 24h 7.2g/L >4.26 0.008 Present study
JAr 10% FCS, pH 7.4, 24h, +cAMP 7.2g/L >4.26 0.005 Present study
JAr 10% FCS, pH 7.4, 72h 7.2g/L >4.26 0.0017 Present study
JAr 10% FCS, pH 7.4, 72h, +cAMP 7.2g/L >4.26 0.0017 Present study
JAr 10% FCS, pH 7.4, 1h, + hCG 7.2g/L na 0.319 Present study
JAr 10% FCS, pH 7.4, 4h, + hCG 7.2g/L na 0.099 Present study
JAr 10% FCS, pH 7.4, 24h, + hCG 7.2g/L na 0.007 Present study
JAr 10% FCS, pH 7.4, 48h, + hCG 7.2g/L na 0.004 Present study
JAr 10% FCS, pH 7.4, 24h 360g/L 16 0.13 Present study
Progesterone secretion by Roundup & Glyphosate exposed cells
MA10 Serum-free, pH na, 2h 180g/L >0.6 0.14 Walsh 2000
JAr 10% FCS, pH 7.4, 24h 7.2g/L >4.26 0.012 Present study
JAr 10% FCS, pH 7.4, 24h, +cAMP 7.2g/L >4.26 0.005 Present study
JAr 10% FCS, pH 7.4, 72h 7.2g/L >4.26 0.007 Present study
JAr 10% FCS, pH 7.4, 72h + cAMP 7.2g/L >4.26 0.008 Present study
JAr 10% FCS, pH 7.4, 24h 360g/L 26 0.2 Present study
JEG and JAr cell lines both derived from human placental choriocarcinoma. HEK- human embryonic kidney cells. MA10 – mouse leydig cell line. FCS – foetal calf serum, >mM – no effect
found at the highest concentration tested. na – information not available.
Table 1. Roundup and Glyphosate EC50 values for cytotoxicity and progesterone synthesis in vitro.
Young F (2015) Endocrine disruption and cytotoxicity of glyphosate and roundup in human JAr cells in vitro
Volume 1(1): 12-19Integr Pharm Toxicol Gentocicol, 2015 doi: 10.15761/IPTG.1000104
Glyphosate (M)
Progesterone (ng/ml) Cells per Well
Controls
Glyphosate
Roundup
Figure 4. Effect of Glyphosate or Roundup (320g/L) on JAr cell viability and
Progesterone Synthesis.
Human JAr cells (40000 per well) were exposed to glyphosate ( ) or Roundup (■,
formulation contained 320g/L glyphosate) in triplicate wells in 96 well plates on 7 separate
occasions (n=7) for 24h. The mean±stdev numbers of surviving cells in each well were
determined in an MTT assay, by comparison with a standard curve generated for each
experimental replicate. The progesterone secreted into the culture media was measured in
an ELISA. Data analysed by 1-way ANOVA with Tukey post-hoc test., and difference from
vehicle control (97% RPMI medium) shown; p<0.05 *, p<0.01 **, p<0.001 ***.
endocrine disruption did not precede cytotoxicity. A 24h exposure
to a concentration of Glyphosate (in Roundup) similar to that
recommended as an acceptable level for Australian drinking water
caused signicant cytotoxicity in vitro, which supports a call for in vivo
studies to characterise the toxicity of Roundup.
Acknowledgement
We are extremely grateful to Dr Alison Bleaney, Dr Marcus
Scammall and Environment Tasmania for funding part of this work.
We are delighted to acknowledge the enthusiastic technical support
provided by the undergraduate and Masters of Biotechnology Studies
students of the Medical Biotechnology BTEC3002/9003 topics at
Flinders University, who exposed the JAr cells to glyphosate or the
320g/L Roundup formulation in vitro, and conducted the MTT assays
and progesterone ELISAs as part of a laboratory practical exercise.
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Copyright: ©2015 Young F. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
... This endocrine disruption altered reproductive behavior, with modifications of sexual preference and a delay in the first mount of females to initiate reproduction (Romano et al. 2012). RoundUp V R could also reduce progesterone production in human placental cells, but only at higher concentrations than ones causing cell death, suggesting that the effect on progesterone is probably related to the cytotoxicity (Young et al. 2015). Human pregnancies were also affected by GBHs, as shown by a correlation between GBH exposure and a higher frequency of miscarriages and pre-term births (Arbuckle et al. 2001). ...
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The anti-HIV agents AZT (zidovudine) and ddI (dideoxyinosine) are being used clinically during pregnancy. The toxicity of these agents to the fetus and placenta remains a concern because few human pregnancy exposure data are available, and pregnant rodent studies with AZT indicate increased embryonic resorptions and developmental arrest. The current study used a human choriocarcinoma cell line (JAr), which exhibits many characteristics of the early placenta, to assess the effects of a single 24 h exposure of 7.6 or 0.076 mM AZT, and the effects of a single 24 h exposure of 7.6 or 0.076 mM ddI upon cell proliferation and hormone production of human chorionic gonadotropin (hCG), estradiol (E2), and progesterone (P4). The higher concentration of AZT and ddI produced significant (P < 0.025) reductions in cell numbers and growth rate while producing significant increases in hormone production (hCG, E2, and P4). The lower concentration of AZT and ddI produced significant increases in E2 production, but no changes in cell numbers, hCG, or P4. Because placental cells require androgen precursor for E2 synthesis, exogenous androstenedione was added to confirm observations of increased estradiol synthesis after AZT or ddI exposure. These results demonstrate that single 24 h high dose exposures of AZT or ddI produce significant inhibition of cell proliferation and alterations in hormone production in this paradigm of human placental cells.