Differential effects of glyphosate and Roundup on
human placental cells and aromatase

SOPHIE RICHARD, et al. Environmental Health Perspectives Online 24feb2005

[More on Roundup and Monsanto]

Sophie Richard, Safa Moslemi, Herbert Sipahutar, Nora Benachour, Gilles-Eric Seralini*
doi:10.1289/ehp.7728 (available at http://dx.doi.org/)

The National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Department of Health and Human Services

Laboratoire de Biochimie, EA2608-USC INRA, IBFA, Esplanade de la Paix, Universit6 de Caen, 14032, FRANCE
*To whom correspondence should be addressed. Tel: 33 (0)2.31.56.54.89, fax: 33 (0)2.31.56.53.20, e-mail: criigen@ibfa.unicaen.fr
Key words: adjuvants, aromatase, endocrine disruption, glyphosate, herbicide, human JEG3 cells, placenta, reductase, Roundup, xenobiotic

Abbreviations: DEAE: Diethyl Amino Ethyl; DTT: dithiothreitol; EMEM: Eagle’s modified Minimum Essential Medium; E1: Estrone; GAPDH: Glyceraldehyde-3-Phosphate Deshydrogenase; IC₅₀: Inhibiting Concentration 50: the concentration of competitor that reduces enzyme velocity by half; Ki: Inhibiting Constant: the dissociation constant of the inhibitor for the enzyme, which has the dimension of a concentration; LD₅₀: Lethal Dose 50: the dose that is lethal to 50% of the exposed cells; M-MLV-RT: Moloney Murine Leukemia Virus Reverse Transcriptase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NADPH: Nicotinamide Adenosine Dinucleotide Phosphate (H⁺: oxidized form); PBS: Phosphate Buffer Saline; PCR: Polymerase Chain Reaction; RIA: Radioimmunoassay; RT: Reverse Transcripted; S.E.M.: Standard Error of the Mean

Acknowledgments: We thank the Quality and Sustainable Development department of Carrefour Group, La Fondation pour une Terre Humaine, CRII-GEN, Ad.Gene laboratory, La Ligue Nationale contre le Cancer, Comite du Calvados, for financial support of the study and a student grant to S.R, SFERE for a student grant to H.S., Marie-Josephe Simon for technical assistance and Frederique Baudoin for secretarial assistance. A conflict of interest was not reported.

Roundup is a glyphosate-based herbicide used worldwide including on most genetically modified plants in which it can be tolerated. Its residues may thus enter the food chain and glyphosate is found as a contaminant in rivers. Some agricultural workers using glyphosate have pregnancy problems, but its mechanism of action in mammals is questioned. Here we show that glyphosate is toxic on human placental JEG3 cells within 18 hr with concentrations lower than the agricultural use, and this effect increases with concentration and time, or in the presence of Roundup adjuvants. Surprisingly, Roundup is always more toxic than its active ingredient. We tested its effect on aromatase with lower non-toxic concentrations, the enzyme responsible for estrogen synthesis. The herbicide acts as an endocrine disruptor on aromatase activity and mRNA levels, and glyphosate interacts within the active site of the purified enzyme, but its effect is facilitated by Roundup formulation in microsomes or in cell culture. We conclude that endocrine and toxic effects of Roundup and not only glyphosate can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances glyphosate bioavailability and / or bioaccumulation.

Glyphosate is known as the active ingredient of the broad-spectrum herbicide Roundup; it inhibits the shikimic acid pathway which is important for plant protein synthesis (Schönbrunn et al. 2001), but it has also been shown to modulate a plant cytochrome P450 (Lamb et al. 1998). It is believed to be rather specific, and less toxic on the ecosystem than other pesticides; transgenic plants tolerant to this compound have even been developed with this argument (Vollenhofer et al. 1999; Williams et al. 2000). However, mammals and humans may be exposed to its residues by agricultural practices (Acquavella et al. 2004) or when they enter the food chain (Takahashi et al. 2001); glyphosate is also found as a contaminant in rivers (Cox 1998). Roundup contains acid glyphosate and adjuvants such as polyethoxylated tallowamine (Cox 1998). Its adjuvants are generally considered as dilutants for regulatory purposes. Although some agricultural workers using glyphosate-based herbicides are reported to have pregnancy problems (Savitz et al. 2000), its mechanism of action in mammals is still questioned and it may have several enzymatic effects (Daruich et al. 2001; Williams et al. 2000). It has also been recently shown to disrupt the animal cell cycle in urchin eggs (Marc et al. 2002), and even the post-transcriptional expression of the steroidogenic acute regulatory protein in mouse testicular Leydig cells (Walsh et al. 2000).

Here we tested glyphosate and Roundup toxicity on human placental JEG3 cells; but also we evaluated its possible capacity to act as an endocrine disruptor like other pesticides (Nativelle-Serpentini et al. 2003), by measuring its effect at non-toxic levels on aromatase, a mammalian cytochrome P450 crucial for sex steroid hormone synthesis. The cytochrome P450 superfamily includes numerous proteins able to metabolize xenobiotics (Nelson 1998). The enzyme aromatase is composed of the product of the CYP19 gene (Bulun et al. 2003) and the associated NADPH-dependent reductase, and is responsible for the irreversible conversion of androgens into estrogens. It is considered as a limiting factor involved in estrogen synthesis and thus in physiological functions including female and male gametogenesis (Carreau 2001), reproduction, sex differentiation or even in bone growth. It is also pharmacologically controlled in estrogen-dependent cancers (Seralini and Moslemi 2001).

The direct action of glyphosate on aromatase could explain some effects on reproduction observed in vivo at least in part, thus we also tested glyphosate and Roundup directly on aromatase present in microsomes from human placenta and equine testis, a tissue known to be aromatase-rich (Lemazurier et al. 2001). We also purified aromatase from this source in order to assess the specificity of the interaction within the active site in this very well characterized mammalian model (Auvray et al. 1998).

N-(Phosphonomethyl) glycine (glyphosate) was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) and the pesticide Roundup (containing 360 g/l acid glyphosate, Monsanto, France) was from a commercial source. A 2% solution of Roundup and an equivalent solution of glyphosate were prepared in Eagle’s modified Minimum Essential Medium (EMEM, Abcys, Paris, France) and the pH of glyphosate solution was adjusted to the pH of the 2% Roundup solution, i.e. about pH 5.8. Successive dilutions were then obtained with serum-free EMEM.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was obtained from Sigma-Aldrich. It was prepared as a 5 mg/ml stock solution in PBS, filtered through a 0.22 µm before use and diluted to 1 mg/ml in serum-free EMEM.

The polyclonal rabbit antibody directed against estrone was purchased from PARIS company (Compiegne, France). Tritiated estrone ([2, 4, 6, 7 ³H] E₁, 95 Ci/mmol, 3,52 TBq) was from Dupont NEN (Les Ulis, France).

The human choriocarcinoma-derived placental JEG3 cell line (ECACC 92120308) was provided by CERDIC (Sophia-Antipolis, France). Cells were grown in phenol red-free EMEM containing 2 mM glutamine, 1% non-essential amino acid, 100 U/ml of antibiotics (mix of penicillin, streptomycin and fungizone), 1 mM sodium pyruvate and 10% fetal calf serum (Biowhittaker, Gagny, France). Fifty thousand cells per well were grown to 80% confluence in 24 well-plates, washed with serum-free EMEM and then were exposed to various concentrations of Roundup or the equivalent concentrations of glyphosate in serum-free EMEM for 1 hr or 18 hr or in serum-containing medium for longer exposures.

This enzymatic test, based on the cleavage of MTT into a blue coloured product (formazan) by mitochondrial enzyme succinate-dehydrogenase (Mossman 1983), was used to evaluate JEG3 cell viability exposed to Roundup or glyphosate during various times. Cells were washed with serum-free EMEM and incubated with 250 µl MTT per well. The plates were incubated for 3 hr at 37°C and 250 µl of 0.04 N hydrochloric acid-containing isopropanol solution were added to each well. The plates were vigorously shaken in order to solubilize the blue formazan crystals formed. The optical density was measured using a spectrophotometer (Stratagene, Strasbourg, France) at 560 nm for test and 640 nm for reference.

The conversion of androstenedione to estrone by the aromatase complex was measured in cell supernatants by radioimmunoassay (RIA) as previously described (Nativelle-Serpentini et al. 2003). JEG3 cells, exposed to Roundup or glyphosate, were washed with serum-free EMEM and incubated for 90 min with 200 nM androstenedione at 37°C in 5% CO₂. The reaction was stopped by placing the plates on ice for 5 min and supernatants were extracted by adding 10 volumes of diethyl ether. The extraction efficiency, evaluated by adding radiolabelled estrone, was 60 ± 3%. The rabbit E1 antibody was prepared according to the manufacturer’s instructions. The sensitivity of the RIA was 10 pg/ml. Intra- and inter-assay coefficients of variation were 4 and 6% respectively. The aromatase activity was expressed in relation to the protein concentration that was evaluated in cell extracts using bovine serum albumin as standard (Bradford 1976).

Total RNA was isolated from JEG3 cells using the guanidium/phenol/chloroform method (Chomczynski and Sacchi 1987). RNA samples were treated with DNase I at 37°C for 30 min to remove genomic DNA. Then DNase I was inactivated at 65°C for 10 min.

Total RNA (1 µg) was reverse-transcribed using 100 U of M-MLV-RT at 42°C for 45 min in the presence of 0.5 µg of 18 mer oligo(dT), 500 µM of each dNTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT and 6 U RNasin in a total volume of 25 µl. The absence of DNA contamination in the RNA samples was checked in controls without M-MLV-RT.

For each run, a master-mix was prepared with 1 X SYBR Green buffer containing 5 mM MgCl2, 200 mM dATP, dCTP, dGTP, 400 mM dUTP, 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Courtaboeuf, France) and 300 nM of each primer: EXIIc sense primer, 5’ TGA GGT CAA GGA ACA CAA GA 3’, exon II-specific (position 9-28) and EXIII antisense primer, 5’ ATC CAC AGG AAT CTG CCG TG 3’, for exon III (position 211-230) (Corbin et al. 1988). Five microliters of each diluted RT sample were added to 20 µl of the PCR master-mix. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 1 min. We also quantified the transcripts of the housekeeping gene, glyceraldehyde-3-phosphate deshydrogenase (GAPDH), as an endogenous control to normalize each sample using sense and antisense primers: 5’ CCA TCA CCA TCT TCC AGG AGC 3’ (position 278-298) and 5’ GGA TGA TGT TCT GGA GAG CC 3’ (position 663-682), respectively (Tokunaga et al. 1987). All PCR reactions were performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems).

Microsomal fractions (endoplasmic reticulum) were obtained from full-term placentas of young healthy and non-smoking women (CHR Caen) and equine testis by differential centrifugations (Moslemi et al. 1997). Briefly, tissues were washed with 0.5 M KCl, homogenized in 50 mM phosphate buffer pH 7.4 containing 0.25 M sucrose and 1 mM dithiothreitol (DTT), and centrifuged at 20,000g. The supernatant was then ultracentrifuged at 100,000g, and the final pellet was washed twice and finally dissolved in the same buffer containing 20% glycerol and stored at –70°C until use. All steps of the preparation were carried out at 4°C.

Microsomal aromatase activity was evaluated by tritiated water release from radiolabelled substrate [1ß-³H] androstenedione as previously described (Moslemi et al. 1993; 1997). This method is based on the stereo-specific release of 1ß-hydrogen from the androstenedione substrate, which forms tritiated water during aromatization (Dintinger et al. 1989; Thompson and Siiteri 1974). Human placental microsomes (50 µg proteins) were incubated with radiolabelled androstenedione (100 pmol/tube) at 37°C for 15 min, in the presence or absence of various concentrations of Roundup or glyphosate in 1 ml total volume of 50 mM Tris-maleate buffer pH 7.4. The reaction was started by adding 100 µl of 0.6 mM NADPH, H⁺ and stopped with 1.5 ml chloroform and then centrifuged at 2700g at 4°C for 5 min. After adding 0.5 ml of 7% charcoal / 1.5% dextran T-70 solution into the preparation, the centrifugation was repeated for 10 min. Aromatase activity was determined by measuring the radioactivity of 0.5 ml aqueous phase. The kinetic parameters were determined by incubating equine testicular microsomes (2 µg proteins) with various concentrations of radiolabelled androstenedione in the presence of various concentrations of Roundup in 0.5 ml NADPH, H⁺ containing-Tris-maleate buffer, pH 7.4 at 25°C for 3 min.

Reductase was obtained after chromatographic separation, by w-aminohexyl-Sepharose 4B and adenosine 2’-5’-diphosphate-agarose respectively, hydrophobic interaction and affinity columns (Vibet et al. 1990). The cytochrome P450 aromatase was purified from equine microsomes, following its separation from reductase, by successive chromatographic steps: concanavalin A-Sepharose 4B affinity column, DEAE-Sepharose CL-6B ion exchange and hydroxyapatite-Sepharose 4B adsorption/partition columns (Moslemi et al. 1997). Protein concentration was determined as previously described (Bradford 1976)

Reductase activity was determined by the measurement of the increasing absorbance of the preparation, corresponding to the reduction of the cytochrome C in the presence of NADPH, H⁺  (Vibet et al. 1990) at 550 nm for 2 min at 37°C using a Kontron-Uvikon 860 spectrophotometer. The pH of the preparation was adjusted when ajusted to 7.4 by adding an appropriate volume of 10 N NaOH. After equilibration, the reaction was started by adding cytochrome C.

The absorbance of purified equine aromatase in the presence or absence of glyphosate or Roundup was recorded from 375 to 475 nm with a spectrophotometer as previously described (Moslemi and Seralini 1997). Briefly, absorption spectra of 362 µg protein of aromatase in 1.5 ml of 50 mM Tris-maleate containing 2 µM androstenedione were recorded during incubation at 37°C, after adding 0.0046% glyphosate or 0.1% Roundup. The spectra of aromatase or glyphosate or Roundup alone were subtracted from the incubation spectrum.

All data are presented as the mean ± standard error (S.E.M.). The experiments were repeated 3 times in triplicate unless indicated. Statistically significant differences were determined by a student test using significant levels of 0.01 (**) and 0.05 (*).

The recommended agricultural use for Roundup is 1-2% in water, and thus we tested its effect on human placental JEG3 cell viability up to 2% after 18, 24 or 48 hr exposures in serum-containing medium, by the MTT assay in conditions previously described (Nativelle-Serpentini et al. 2003), in comparison to glyphosate. The Roundup dilutions and equivalent quantities of glyphosate were adjusted to the same pH to facilitate the comparisons. The toxicity increased with time (8 times at 0.8% between 24 and 48 h), and the LD₅₀ was approximately 1.8 times lower for Roundup (0.7%) than for glyphosate (Figure 1). This difference was even visible after 1 hr of incubation in serum-free medium (Figure 2A) and increased 3 times after 18 hr of incubation (Figure 2B). Acidity of the 2% Roundup or glyphosate solution (pH 5.80 ± 0.08 instead of pH 7.91 ± 0.16) reduced cell viability only 23% after 18 hr, and thus could not alone explain the 90% reduction of cell viability observed at this concentration. When only 0.1% Roundup was added to glyphosate, bringing little amounts of the adjuvants to the solution, the cell viability was diminished significantly (Figure 2B).

Aromatase activity was measured after incubation of cells in the presence of non-toxic concentrations of Roundup or glyphosate, by radioimmunoassay of estrone formed from 200 nM androstenedione, as previously described (Nativelle-Serpentini et al. 2003). As shown in figure 3A, after 1 hr incubation, the estrogen synthesis was enhanced by about 40% but only with Roundup. After 18 hr incubation a clear inhibition of aromatase activity in vitro was noticed, with an IC₅₀ of 0.04% again with Roundup only. This inhibition of aromatase activity is, at least in part, assumed by an effect on aromatase gene expression since mRNA levels were decreased (Figure 3B). Glyphosate was inefficient alone in these conditions. But it inhibited aromatase activity with minute dilutions of Roundup, bringing adjuvants in the solution (Figure 4).

Microsomal aromatase activity was evaluated by tritiated water release from the radiolabelled substrate (Dintinger et al. 1989; Thompson and Siiteri, 1974) in human (Fig. 5) and equine microsomes. Aromatase inhibition by Roundup was equivalent in these two mammalian models. The IC₅₀ was 0.6% for Roundup in these conditions and more than 3 times greater for glyphosate. The kinetic parameters were determined by incubating equine testicular microsomes with various concentrations of radiolabelled androstenedione and Roundup. The Ki (0.6%) showed a competitive inhibition (Figure 6A).

We further purified the enzyme moieties from the aromatase-rich equine testis, giving better yields than placenta. The incubation with the herbicide demonstrated a direct interaction of glyphosate within the active site. Spectral interactions between Roundup or glyphosate and the active site of the purified cytochrome P450 aromatase were obtained by measuring the absorbance of the preparations from 375 to 475 nm. A type II spectrum was observed (Figure 6B); it was characteristic of an interaction between a nitrogen atom of the molecule and the heme iron of the cytochrome. In addition, we tested the effect of the herbicide on the ubiquitous moiety of the aromatase, which is the electron donor reductase. NADPH-dependent reductase activity was determined by the measurement of the increasing absorbance of the preparation, corresponding to the reduction of the cytochrome C. Reductase is also directly affected after purification and incubation with Roundup but to a lesser extent (IC₅₀ 5%) than the cytochrome P450 aromatase responsible for steroid binding and catalysis (Figure 7).

This study demonstrates that Roundup reduces JEG3 cell viability at least 2 times more efficiently than glyphosate. This effect increased with time and was obtained with concentrations of Roundup 10 times lower than the agricultural use. The presence of serum buffers the toxic effect of the herbicide. It is generally recognized that serum proteins can bind to chemicals and reduce their availability to cells. Seibert et al. (2002) have shown that the presence of albumin influences the cytotoxicity of compounds. Moreover, the lack of growth factors in serum-free medium, for instance, could also play a role in this phenomenon. In our experiments, the incubation in serum-free medium was interesting to optimise the visible effects of the compounds in the shortest time. These were also observed anyway after 48 hr in the presence of serum. The physiological significance of these effects can be questioned, in regard to the concentration used. However the time of exposure to pollutants may be longer in vivo; and here in vitro we observed that long times of exposure allowed low concentrations to present toxic effects. This phenomenon could be due to metabolism, genomic action and / or bioaccumulation of some products of Roundup. For instance Peluso et al. (1998) demonstrated the formation of covalent links between DNA and some Roundup adjuvants. Their genotoxicity or toxicity was also noticed (Lioi et al. 1998; Mitchell et al. 1987; Vigfusson and Vyse 1980). Even though absorbed Roundup is excreted rapidly from the body, usually in feces (Brewster et al. 1991; Williams et al. 2000), a part may be retained or conjugated with other compounds that can stimulate biochemical and physiological responses. The bioaccumulation of some of its residues may be hypothesized. For example, the harmful effect of glyphosate on semen quality after 6 weeks of post-treatment period in rabbits (Yousef et al. 1995) may be considered as an indication of its retention and conjugation in the body, helped by Roundup adjuvants.

Additionally, in this work Roundup presents a differential time effect at non-toxic levels on aromatase activity of JEG3 cells, this phenomenon was already observed with other xenobiotics like lindane and bisphenol-A (Nativelle-Serpentini et al. 2003). The 40% rise in aromatase activity after 1 hr of incubation is perhaps due to an increase of the membrane fluidity and androgenic substrate bioavailability in a first step provoked by adjuvants. By contrast, once well entered into cells, Roundup always reduced aromatase activity. Furthermore, this was associated with the decrease of CYP19 mRNAs. Walsh et al. (2000) showed that Roundup preferentially diminished the expression of StAR mRNA by decreasing at least the rate of gene transcription.

The direct inhibition of aromatase activity by Roundup was verified in human and equine microsomes, two mammalian aromatase models that we have precisely characterized, in order to understand the active site configuration of this membrane bound cytochrome P450 (Auvray et al. 1998; Moslemi and Seralini 1997; Seralini et al. 2003). Contrary to results obtained in cells, glyphosate had an inhibitory effect on aromatase activity in human and equine microsomes, but 4 times lower than the effects of Roundup. Moreover Roundup inhibited aromatase better in cells than in microsomes (IC₅₀ 0.04 and 0.6% respectively). This could be explained by the difference in incubation duration (18 h in comparison to 15 min) inducing metabolism and genomic action. Glyphosate penetration through the cell membrane and subsequent intracellular action appeared in our work to be greatly facilitated by adjuvants, like in plants (Haefs et al., 2002) or in animal cells, where it can act at the level of cycle regulation (Marc et al. 2002). Indeed, in this work, minute dilutions of Roundup bringing adjuvants to cells allowed the aromatase inhibitory effect of glyphosate as well as cytotoxic effects.

Moreover, the presence of Roundup in the incubation medium resulted not only in the decrease of the activity of the cytochrome P450 aromatase, but also to a lesser extent in a partial inhibition of its associated reductase. This is confirmed by kinetic and spectral studies which showed that Roundup inhibits the enzyme at the active site level in a competitive manner. Furthermore, our spectral study shows a type-II spectrum for purified equine aromatase in the presence of glyphosate or Roundup at the saturating concentration of androstenedione. After androstenedione elimination, Roundup induces a type-I spectrum. A type-II spectrum with minimal absorbance at 390 nm and maximal absorbance at 420 nm is considered as specific for an interaction between a nitrogen atom of the molecule and the heme iron of the cytochrome, whereas a type-I spectrum (inverted absorbance) is observed when this type of interaction is absent. Androstenedione, a natural hormone, thus appears to facilitate pesticide access to the active site of the enzyme. However, this occurs more easily with glyphosate directly in contact with the solubilized enzyme than with Roundup, since less concentration of the former was needed to produce the same spectrum.

Our studies show that glyphosate acts as a disruptor of mammalian cytochrome P450 aromatase activity from concentrations 100 times lower than the recommended use in agriculture, and this is noticeable on human placental cells after only 18 hr, and it can also affect aromatase gene expression. It also partially disrupts the ubiquitous reductase activity but at higher concentrations. Its effects are allowed and amplified by at least 0.02% of the adjuvants present in Roundup, known to facilitate cell penetration, and this should be carefully taken into account in pesticide evaluation. The dilution of glyphosate in Roundup formulation may multiply its endocrine effect. Roundup may be thus considered as a potential endocrine disruptor. Moreover, at higher doses still below the classical agricultural dilutions, its toxicity on placental cells could favor some reproduction problems.

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Figure 1. Effects of Roundup (A) and equivalent quantities of glyphosate (B) on JEG3 placental cell viability in a serum-containing medium. This was evaluated by the MTT assay, the results are presented in % comparatively to non-treated cells. Cells were incubated with increasing concentrations of Roundup or equivalent concentrations of glyphosate for 18, 24 or 48 hr (n = 9). The LD₅₀ is indicated by a dotted line. For all figures, S.E.M. are shown, *p<0.05; **p<0.01.

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Figure 2. Effects of Roundup and equivalent quantities of glyphosate on JEG3 placental cell viability in serum-free medium. The incubation was for 1 hr (A) or 18 hr (B). The addition of 0.02 or 0.1% Roundup shows adjuvants effects (n = 9).

Figure 3. Effects of Roundup and equivalent quantities of glyphosate on JEG3 aromatase activity and mRNA levels in a serum-free medium. Aromatase activity (A) was obtained with non-toxic concentrations of Roundup or glyphosate for 1 and 18 hr (n = 9). Cytochrome P450 aromatase mRNA levels, normalized with GAPDH ones (B), in the presence of Roundup or glyphosate after 18 hr (n = 4).

Figure 4. Combined effects of glyphosate and minute levels of Roundup on JEG3 aromatase activity in serum-free medium (n = 9). It was obtained at non-toxic concentrations after 18 hr exposure.

Figure 5. Effects of Roundup and equivalent quantities of glyphosate on microsomal aromatase activity. Human placental microsomes were incubated with Roundup or glyphosate at 37°C for 15 min (n = 9). The IC₅₀ is indicated by a dotted line. Similar results were obtained with equine testicular microsomes.

Figure 6. Kinetic and spectral studies of aromatase in the presence of Roundup or glyphosate. (A) Lineweaver-Burk representation of equine testicular microsomal aromatase activity in the presence of Roundup at 25°C with radiolabelled androstenedione. Comparable results were obtained with human placental microsomes (n = 9). (B) Spectral analysis of interactions between the active site of purified equine cytochrome P450 aromatase and 0.1% Roundup (left) or 0.0045% glyphosate (right). Type II spectra were obtained with Roundup or glyphosate in the presence of 2 µM androstenedione and a type I spectrum was obtained in its absence. The results are representative of 3 experiments.

Figure 7. Effect of Roundup on reductase activity. Activity of purified equine reductase was measured in the presence of increasing concentrations of Roundup in non-adjusted or adjusted pH (7.4) medium for 15 min at 37°C (n = 9).