Aromatase disruption

Aromatase activity was evaluated according to the tritiated water release assay

(Thompson and Siiteri, 1974) with a slight modification as previously described

(Dintinger et al., 1989). This method is based on the stereo-specific release of

1 -hydrogen from the androstenedione substrate, which forms tritiated water during

aromatization. The HepG2 cells were exposed to non-toxic concentrations of

glyphosate alone or Roundup, and were washed with serum-free EMEM and incubated

for 90 min with 200 nM [1 -3H] androstenedione at 37 ◦C (5% CO2, 95% air).

The reaction was stopped by centrifugation at 2700×g at 4 ◦C for 10 min. After

adding 0.5mL of charcoal/dextran T-70 suspension, the mixture was centrifuged

similarly. Supernatant fractions were assessed for radioactivity by scintillation

counting (Parckard, Liquid scintillation counter 1600LR, USA).

Aromatase mRNA levelswere measured by semi-quantitative RT-PCR. Total RNA

was extracted (RNAgentsmethod, Promega, F) from HepG2 cells and checked at 260,

280nmand by electrophoresis on agarose gel (1.5%) stainedwith ethidium bromide.

Five micron were reverse-transcribed (RT) using 200U MMLV-RT (Moloney murine

leukemia virus reverse transcriptase) at 42 ◦C for 60 min in the presence of 0.2 g

oligo dT, 500 Mof each dNTP and 20U RNasin in a total volume of 40 L. The cDNA

obtained were used for PCR. For each run, a master mix was prepared with 1.5 IU

Taq DNA polymerase in PCR buffer containing 200mM dNTP, 1.5mM MgCl2, and

25 pmoles of each primer in a total volume of 50 L. The PCR primers were EXIIc

sense, 5 TGA GGT CAA GGA ACA CAA GA 3 and EXIII antisense 5 ATC CAC AGG

AAT CTG CCG TG 3 (Corbin et al., 1988). The thermal cycling conditions consisted

of an initial step at 95 ◦C for 2min and then 35 cycles of 95 ◦C for 30 s and 60 ◦C

for 60 s. Aromatase mRNA levels were normalized with the control housekeeping

gene GAPDH. The primers used for PCR was for the sense primers 5 CCA TCA CCA

TCT TCC AGG AGC 3 and for the antisense 5 GGA TGA TGT TCT GGA GAG CC 3 . The

resulting PCR productswere analyzed by electrophoresis on a 2% agarose gel stained

with ethidium bromide. Gels were photographed using photoprint Vilbert Lourmat

(F) system and analyzed with image J computer program.

 Anti-estrogenic activity

Five plasmids were used for the transient transfections of the HepG2 cell line.

Plasmids ERE-TK-Luc, hER and hER were kindly provided by Dr D. McDonnell

(Ligand Pharmaceutical, San Diego, USA); pCMV Gal and psG5 were used for the

normalization of luciferase activity (Cabaton et al., 2009). ERE-TK-Luc is a 6.7 kb

expression vector containing a single copy of the estrogen response element of the

vitellogenin with a minimal thymidine kinase promoter driving firefly luciferase

(Tzukerman et al., 1994). Plasmids hER and hER are built from the plasmid pRSTER

(Rous Sarcoma Virus/T7 promoter; Hall and McDonnell, 1999) and encode the

human wild-type estrogen receptor or . The pCMV Gal contains -galactosidase

gene and is used in order to control the transfection efficiency. Finally, pSG5 is used

to obtain an appropriate DNA concentration for the transfection.

HepG2 cells were transiently transfected using Exgen 500 procedure

(Euromedex, Mundolsheim, F). 120,000 cells per well were grown at 37 ◦C (5% CO2,

95% air) in MEM supplemented with 2mM glutamine, 1% non-essential aminoacid

and 10% of dextran-coated charcoal fetal calf serum in 24-well plates. The

microplates were then incubated for 24 h. For transfections, all plasmids were first

diluted in 0.15M NaCl to a final concentration of 100 ng/ L and then mixed: 100 ng

ERE-TK-Luc, 100 ng hER or , 100 ng pCMV Gal and 200 ng pSG5. Then 2 L of

Exgen 500 diluted in NaCL 0.15Mwere added to DNA. The mix was centrifuged and

incubated at least 10min at room temperature. The mixturewas added to OptiMEM

and distributed into the wells (300 L/well). After 1 h of incubation (37 ◦C, 5% CO2),

the medium was removed and replaced by 1mL of treatment medium without fetal

calf serum for 24 h. To observe an anti-estrogenic activity, cellswere co-treated with

xenobiotics and 17 -estradiol 10−8 M. ICI 182×780 (10−8 M) was used as positive

control. At the end of the treatment, cells were lysed with Reporter Lysis Buffer

(Promega) and frozen at −80 ◦C for at least 30min. Then they were scraped and

placed intomicrotubes before three freezing (liquid nitrogen)/thawing (37 ◦C water

bath) cycles and centrifuged 5min at 10,600×g.

For luciferase activity measurement, 10 L of lysat were mixed with 50 L

of luciferase assay reagent (Promega) into a white 96-well plate. The mixtures

were immediately analysed using a luminometer (TopCount NT, Packard). The -

Galactosidase activitywasmeasuredusing chlorophenol-red -d-galactopyranoside

(Roche Diagnostics GmbH, Mannheim, Germany). The chlorophenol-red product

was measured with a spectrophotometer at 570nm (MRX Dynex). Protein concentration

determination was performed using 2 L of the lysate according to Bradford

(1976) on a spectrophotometer at 595 nm. Luciferase activity for each treatment

group was normalized to galactosidase activity and protein level (Luc×Prot/Gal)

was compared to the control (17 -estradiol 10−8 M) set at 100%.

 Anti-androgenic activity

MDA-MB-453-kb2 cells were seeded in 24-well plates and 50,000 cells per well

were grown in L-15 medium without phenol-red supplemented with 5% dextrancharcoal

fetal calf serum for 24 h (37 ◦C without CO2). After 24 h incubation, medium

was removed and cells were washed with 500 L PBS and exposed to roundup

solutions in co-treatment with DHT (4×10−10 M) for 24 h in medium without fetal

calf serum. Nilutamide (10−6 M) was used as positive control. For luciferase activity

measurement, 10 L of lysate were mixed with 40 L of luciferase assay system

(Promega) into awhite96-well plate. The mixtureswereimmediately analysedusing

a luminometer (TopCount NT, Packard). Results were expressed as a percentage of

the data obtained with the androgen DHT (4×10−10 M).

Fig. 1. Dose-dependent effects of glyphosate (G) and four glyphosate-based formulations (Roundup containing 7.2–450 g/L G) on HepG2 cells viability after 24 h of exposure.

These effects were evaluated by the MTT test (A) or the ToxiLight assay (B). The results are presented in % comparatively to non-treated cells (100% viability, A) or in relative

levels to non-treated cells (URL: 1, B). Cells were grown at 37 ◦C (5% CO2, 95% air) in medium EMEM with 10% serum during 48 h to 80% confluence in 48-well plates for MTT

test or 96-well plates for ToxiLight, and then exposed to the products for 24 h without serum. All experiments were repeated 4 times in triplicates.