martes, 17 de agosto de 2010
Glyphosate- based herbicides produce teratogenic effects on vertebrates by impairing retinoic Acid Signaling
Glyphosate-Based Herbicides Produce Teratogenic Effects on Vertebrates
by Impairing Retinoic Acid Signaling
Alejandra Paganelli, Victoria Gnazzo, Helena Acosta, Silvia L. López, andAndrés E. Carrasco*
Laboratorio de Embriologıá Molecular, CONICET-UBA,
Facultad de Medicina,
UniVersidad de Buenos Aires,Paraguay 2155, 3° piso (1121),
Ciudad Autónoma de Buenos Aires, Argentina
ReceiVed May 20, 2010
The broad spectrum herbicide glyphosate is widely used in agriculture worldwide. There has been on going controversy regarding the possible adverse effects of glyphosate on the environment and onhuman health. Reports of neural defects and craniofacial malformations from regions where glyphosate-based herbicides (GBH) are used led us to undertake an embryological approach to explore the effectsof low doses of glyphosate in development. Xenopus laeVis embryos were incubated with 1/5000 dilutionsof a commercial GBH. The treated embryos were highly abnormal with marked alterations in cephalicand neural crest development and shortening of the anterior-posterior (A-P) axis. Alterations on neuralcrest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryosinjected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effectsin chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, andmicrocephaly. This suggests that glyphosate itself was responsible for the phenotypes observed, ratherthan a surfactant or other component of the commercial formulation. A reporter gene assay revealed thatGBH treatment increased endogenous retinoic acid (RA) activity in Xenopus embryos and cotreatmentwith a RA antagonist rescued the teratogenic effects of the GBH. Therefore, we conclude that thephenotypes produced by GBH are mainly a consequence of the increase of endogenous retinoid activity.This is consistent with the decrease of Sonic hedgehog (Shh) signaling from the embryonic dorsal midline,with the inhibition of otx2 expression and with the disruption of cephalic neural crest development. Thedirect effect of glyphosate on early mechanisms of morphogenesis in vertebrate embryos opens concernsabout the clinical findings from human offspring in populations exposed to GBH in agricultural fields.IntroductionThe broad-spectrum glyphosate based herbicides (GBHs) arewidely used in agricultural practice, particularly in associationwith genetically modified organisms (GMO) engineered to beglyphosate resistant such as soy crops. Considering the wideuse of GBH/GMO agriculture, studies of the possible impactsof GBH on environmental and human health are timely andimportant. Given the intensive use of this technological packagein South America, studies of the possible impacts on environ-ment and human health are absolutely necessary, together withadequate epidemiological studies. The need for informationabout the developmental impact of GBH is reinforced by avariety of adverse health effects on people living in areas whereGBH is extensively used, particularly since there is a paucityof data regarding chronic exposure to sublethal doses duringembryonic development.It is important to note that the bulk of the data provided duringthe evaluation stages of GBH/GMO safety were provided bythe industry. Given the recent history of the endocrine disruptorfield with low dose effects observed in numerous academiclaboratories but not in industry-funded studies (1, 2), it is clearthat a reasonable corpus of independent studies is necessary tofully evaluate the effects of agrochemicals on human health.This is particularly important when significant economicinterests are concerned.There is growing evidence raising concerns about the effectsof GBH on people living in areas where herbicides areintensively used. Women exposed during pregnancy to herbi-cides delivered offspring with congenital malformations, includ-ing microcephaly, anencephaly, and cranial malformations (3).Relevant contributions to the subject were made by Seralini’sgroup, among others (4). They showed that a GBH acts as anendocrine disruptor in cultures of JEG3 placental cells, decreas-ing the mRNA levels of the enzyme CYP19 (an essentialcomponent of cytochrome p450 aromatase) and inhibiting itsactivity. CYP19 is responsible for the irreversible conversionof androgens into estrogens. The GBH Roundup is able todisrupt aromatase activity. Importantly, the active principleglyphosate interacts with the active site of the purified enzymeand its effects in cell cultures, and microsomes are facilitatedby other components in the Roundup formulation that presum-ably increase the bioavailability of glyphosate (4). Glyphosatepenetration through the cell membrane and subsequent intra-cellular action is greatly facilitated by adjuvants such assurfactants (5, 6).In addition, both glyphosate and the commercial herbicideseverely affect embryonic and placental cells, producing mito-chondrial damage, necrosis, and programmed cell death by theactivation of caspases 3/7 in cell culture within 24 h with dosesfar below those used in agriculture. Other effects observedinclude cytotoxicity and genotoxicity, endocrine disruption ofthe androgen and estrogen receptors, and DNA damage in celllines (7, 8).* Corresponding author. Phone:+5411 5950 9500 ext. 2216. Fax:+54115950 9626. E-mail: firstname.lastname@example.org.Chem. Res. Toxicol. XXXX, xxx, 000A10.1021/tx1001749 XXXX American Chemical Society
More recently, rats fed with a glyphosate-resistant geneticallymodified corn showed functional alterations in two detoxificantsorgans: kidney and liver, and the heart and the hematopoieticsystem (9).Another line of evidence supporting adverse effects ofglyphosate was provided by Bellé’s group. They suggested thatglyphosate and its principal metabolite, AMPA, alter cell cyclecheckpoints by interfering with the physiological DNA repairmachinery. Several GBHs were assayed, and they induced cell-cycle dysfunction from the first cell division in sea urchinembryos (10, 11). The threshold concentration for this effect is500- to 4000-fold lower than that sprayed on crops in the field.Eight millimolar glyphosate induces a delay in the kinetics ofthe first cell cleavage of sea urchins, altering the entry intoS-phase by interfering with the activation of the CDK1/cyclinB complex (6, 12). This failure of cell-cycle checkpoints isknown to lead to genomic instability and the possible develop-ment of cancer. In agreement with these findings, genotoxicitystudies of glyphosate or its metabolites suggest that theirreversible damage in the DNA may increase the risk ofcarcinogenesis (13, 14).Aside from the previously reported teratogenic effects ofglyphosate-based formulations on cephalic structures in amphib-ians (15), there is almost no information available about themolecular mechanisms associated with GBH or glyphosateteratogenesis. Reports of neural defects and craniofacial mal-formations from regions where GBHs are used heavily led usto an embryological approach to explore the effects of low dosesof glyphosate in Xenopus and chicken embryogenesis.We show here that sublethal doses are sufficient to inducereproducible malformations in Xenopus and chicken embryostreated with a 1/5000 dilution of a GBH formulation (equivalentto 430 µM of glyphosate) or in frog embryos injected withglyphosate alone (between 8 and 12 µM per injected cell). GBHtreated or glyphosate injected frog embryos showed very similarphenotypes, including shortening of the trunk, cephalic reduc-tion, microphthalmy, cyclopia, reduction of the neural crestterritory at neurula stages, and craniofacial malformations attadpole stages. These defects suggested a link with the retinoicacid (RA) signaling pathway. Reporter gene assays using a RA-dependent reporter revealed that GBH treatment increasesendogenous RA activity. Strikingly, we demonstrate that Ro41-5253 (Ro), an antagonist of RA (16, 17), rescues thephenotype produced by GBH. We propose that at least someof the teratogenic effects of GBH are mediated by increasedendogenous RA activity in the embryos. This is consistent withthe very well-known syndrome produced by an excess of RA,as described by the epidemiological study of Lammer et al. inhumans (18) and in vertebrate embryos (19-25).Experimental ProceduresEmbryo Culture and Treatments. Xenopus laeVis embryoswere obtained by in vitro fertilization, incubated in 0.1× modifiedBarth’s saline (MBS) (26) and staged according to Nieuwkoop andFaber (27). The GBH used was Roundup Classic (Monsanto),containing 48% w/v of a glyphosate salt. Treatments wereperformed from the 2-cell stage with GBH dilutions of 1/3000,1/4000, and 1/5000 prepared in 0.1× MBS. For rescue experiments,0.5 or 1 µM Ro-415253 was added at stage 9. Cyclopamine (SigmaC4116) was used at 100 µM concentration in 0.1× MBS and wasapplied from the 2-cell stage until fixation. Embryos were fixed inMEMFA (28) when sibling controls reached the desired stage.Xenopus Embryo Injections, Whole Mount in Situ Hybridiza-tion and Cartilage Staining. Embryos were injected with 360 or500 pg of glyphosate (N-(phosphonomethyl) glycine (Sigma337757) per cell into one or both cells at the 2-cell stage. Glyphosatewas coinjected with 10 ng of Dextran Oregon Green (DOG,Molecular Probes) to identify the injected side as previouslydescribed (29). Embryos were cultured in 0.1× MBS and fixed inMEMFA when sibling controls reached the desired stage. Whole-mount in situ hybridization (WMISH) with digoxigenin-labeledantisense RNA probes was performed as previously described (30)except that the proteinase K step was omitted. For cartilagevisualization, embryos were fixed in MEMFA at stages 45-47,washed with PBS, and stained overnight in 0.04% Alcian blue, 20%acetic acid, and 80% ethanol. After extensive washing with ethanoland bleaching with 2% KOH, embryos were washed with 20%glycerol and 2% KOH, and dehydrated through a glycerol/2% KOHseries until 80% glycerol was reached.Detection of RA Activity. Embryos were injected into one cellat the 2-cell stage with 320 pg of the plasmid RAREhplacZ(RAREZ) (31, 32) and placed immediately in 1/3000, 1/4000, and1/5000 GBH dilutions. Basal luminiscence was detected in unin-jected and untreated embryos. The endogenous RA activity wasmeasured in embryos injected with RAREZ and left untreated. Aspositive controls, embryos were injected with the RAREZ plasmidand incubated at late blastula stage with 0.5 or 5 µM all-trans-retinoic acid (RA, Sigma R2625). For rescue experiments, embryosinjected with the reporter plasmid were incubated in a 1/4000dilution of GBH from the 2-cell stage, and when they reached theblastula stage, 1 µM of Ro 41-5253 was added. Finally, whensibling controls reached the neurula stages (14, 15), all embryoswere processed for chemiluminiscent quantitation of the reporteractivity by using the β-gal reporter gene assay (Roche). Proteinextracts and enzymatic reactions were performed as previouslyreported (33). Luminiscence was measured on duplicate samplesin FlexStation 3 equipment (Molecular Devices), and values werenormalized by protein content (32). A two-tailed t test wasemployed to analyze the significance in the difference of the means.The experiment was repeated three times.Treatments of Chicken Embryos. After opening a smallwindow in the shell, fertilized chicken eggs (White Leghorn strain)were injected above the air chamber in the inner membrane with20 µL of 1/3500 or 1/4500 dilutions of GBH. Control embryoswere injected only with 20 µL of H2O. After injection, the windowwas sealed with transparent adhesive tape, and eggs were placedwith their blunt end up at room temperature for 30 min. Then, eggswere incubated in darkness at 38 °C in a humidified incubator(56-58% humidity) and rotated at regular intervals. After appropri-ate incubation times, embryos were isolated and staged accordingto Hamburger and Hamilton (34).Whole-Mount Inmunofluorescence and WMISH of ChickenEmbryos. Embryos were fixed 2-4 h in freshly prepared 4%paraformaldehyde, rinsed, and processed for analysis. For immu-nofluorescence, embryos were blocked overnight at 4 °C in blockingsolution (5% normal goat serum, 0.3% Triton X-100, 0.01% NaN3,and Tris buffer saline (TBS) at pH 7.4). Then, they were incubatedwith a 1/50 dilution of a mouse anti-Pax6 monoclonal primaryantibody (Developmental Hybridoma Bank) in TBS at pH 7.4 and0.3% Triton X-100 for 48 h at 4 °C. Embryos were washed threetimes with TBS and incubated at 4 °C with the secondary antibody(1/1000 fluorescein-conjugated (FITC) antimouse IgG, JacksonImmunoResearch) in TBS at pH 7.4, 0.3% Triton X-100, and 3%normal goat serum for at least 12 h. Finally, embryos were washedwith TBS, placed in a glass culture dish with 80% v/v of glycerolin water, and photographed. WMISH was performed as describedfor Xenopus embryos, using a c-shh probe.ResultsGBH and Glyphosate Alter Neural Crest Markers, Rhom-bomeric Patterning, and Primary Neuron Differentiation.In order to examine whether GBH treatment can affect neuralcrest development, rhombomeric patterning, and neuronaldifferentiation, 2-cell stage Xenopus laeVis embryos wereB Chem. Res. Toxicol., Vol. xxx, No. xx, XXXXPaganelli et al.
exposed to GBH, as described in Experimental Procedures, andassayed by whole mount in situ hybridization (WMISH) at theneurula stage (stage 14-15). The neural crest marker slug beginsits expression early, where neural crest induction takes place.At neurula stage, it is expressed in the neural crest territory(Figure 1A, arrows) (35). Treated embryos show an importantdown-regulation of slug in the neural crest territory (Figure 1B,arrows) in comparison with that of sibling controls. To studythe effects on hindbrain patterning, we analyzed the expressionof krox-20. This zinc finger transcription factor is expressed inrhombomeres r3 and r5 (Figure 1A) and has been shown toplay an important role in controlling rhombomere identity (36).The r3 stripe was lost in GBH-treated embryos (Figure 1B).This resembles the progressive loss from anterior to posteriorrhombomeres associated with increasing concentrations of RAtreatments in Xenopus and mouse embryos (37, 38).Then we investigated primary neurogenesis at the neural platestage. At this time, N-tubulin is normally expressed in dif-ferentiated primary neurons organized in three longitudinaldomains in the posterior neural plate: medial, intermediate, andlateral, which correspond to motor neurons (m), interneurons(i), and sensory neurons (s), respectively (Figure 1C) (39).Treated embryos showed a down-regulation in the three stripesof primary neurons (Figure 1D).To corroborate if the effect is specifically due to the activeprinciple of the herbicide and not to adjuvants present informulations, glyphosate was injected into one cell at the 2-cellstage and slug, krox-20, and N-tubulin were revealed at stages14-15, as before. These embryos showed an important down-regulation of slug (Figure 1E, arrow), resembling the effects ofGBH on this marker at this stage of development. AlthoughKrox-20 did not completely disappear from r3 as in GBH-treatedembryos, the expression clearly decreased in this rhombomereas well as in r5, indicating that glyphosate also alters rhombo-meric patterning (Figure 1E; arrowheads).Normally, at stage 18, the neural crest has formed threepremigratory blocks from which three different segmentssegregate: mandibular crest segment, hyoid crest segment, andbranchial crest segment (MCS, HCS, and BCS; Figure 1F). Thefirst segment contributes to the Meckel, quadrate, and ethmoid-trabecular cartilages; the hyoid crest segment to the ceratohyalcartilage, and the branchial segment to the cartilages of the gills(40). Glyphosate-injected embryos showed that the segregationprocess clearly affected the injected side (Figure 1F, arrow),suggesting that the derived cartilages may be affected at laterstages during development. When hybridized with N-tubulin,these embryos showed a decrease in the number of primaryneurons in the three stripes corresponding to motor neurons,interneurons, and sensory neurons (Figure 1G, arrows), resem-bling the effects of GBH treatments, although with milderconsequences for this marker.In conclusion, the effects of GBH-treated and glyphosate-injected embryos represent equivalent phenotypes despite thefact that they are not identical. The adjuvant present in thecommercial formulation may explain the differences. Takentogether, these results indicate that both GBH and glyphosateimpair neuronal differentiation, rhombomeric formation, and thepattern of the neural crest during induction and segregation.GBH and Glyphosate Produce Head Defects and Impairthe Expression of Dorsal Midline and Cephalic Markers.Because craniofacial defects were observed in humans residingin areas chronically exposed to GBH, we decided to explorewhether genes involved in head development are altered as aconsequence of treatment with GBH or injection of glyphosate.Shh acts as a morphogen controlling multiple developmentalprocesses. During early vertebrate embryogenesis, shh expressedin midline structures such as the notochord, prechordal meso-derm, and floor plate controls left-right asymmetry, neuronidentity, neural survival, and dorso-ventral patterning of theneural tube (41, 42). Moreover, Shh secreted by the prechordalmesoderm is responsible for resolving the brain and the retinafield into two separate hemispheres and eyes, preventingcyclopia (43).Shh expression was dramatically reduced in the dorsal midlineat neurula stages, especially in the prechordal mesoderm inGBH-treated embryos. The anterior limit of the shh expressiondomain is moved caudally in treated embryos, in relation tothe pax6 domain (compare green arrowheads, Figure 2A-C).Pax6 is essential for eye formation in a wide range of species.It is expressed in the eye primordia of vertebrates such as themouse, chicken, Xenopus, zebrafish, and humans, as well as ininvertebrates such as Drosophila (44-47). Embryos incubatedwith GBH showed a distinct down-regulation of the pax6territory (compare white arrowheads; Figure 2A-C). Moreover,in treated embryos, the pax6 domain is not divided in the eyefield (light blue arrowheads; Figure 2B,C). These results suggestFigure 1. GBH and glyphosate disturb neural crest formation, rhom-bomeric patterning and primary neuron differentiation. (A-G) Embryoswere analyzed at neurula stage by WMISH with different markers. Allare dorsal views (anterior is up). (A,C) Control embryos. (B,D) Embryostreated with 1/5000 dilution of GBH. (B) Impairment of neural crestformation as revealed by the specific marker slug (arrows). Notice thedown-regulation of the krox-20 domain in the r3 rhombomere. Slugand krox-20 were down-regulated in 87% of treated embryos (n)30).(D) Suppression of primary neuron formation as seen with thedifferentiation marker N-tubulin. The number of primary neurons wasdecreased in 83% of treated embryos (n)30). (E-G) Embryosunilaterally injected with 500 pg of glyphosate per cell plus DOG asthe tracer. The injected side is demarcated by the green fluorescencein the insets and is oriented to the left. IS, injected side. NIS, noninjectedside. (E,F) Abolishment of slug expression in the cranial neural crestdomains (arrow; 77%, n)31) and diminution of krox-20 expressionin r3 and r5 (arrowheads; 71%, n)21) on the IS. (G) Reduction ofN-tubulin expression on the IS (81%, n)16). r3, is the thirdrhombomere; r5, fifth rhombomere. m, i, and s, are primary motorneurons, interneurons, and sensory neurons, respectively. MCS, HCS,and BCS, are mandibular crest segment, hyoid crest segment, andbranchial crest segment, respectively.Teratogenic Effects of GlyphosateChem. Res. Toxicol., Vol. xxx, No. xx, XXXX C
that a down-regulation of shh expression in the prechordalmesoderm together with a diminution of pax6 expression mayunderlie the defects in the resolution of the retina field and thebrain hemispheres in embryos treated with GBH.To test whether these molecular alterations were associatedwith defects at later stages, we analyzed the expression of shh/otx2 (Figure 2D,F) and sox9 (Figure 2E,G) in embryos treatedwith GBH as before but fixed at tailbud stages. Otx2 is ahomeobox-containing gene expressed in retinal and lens com-ponents of the eye and telencephalic, diencephalic, and mes-encephalic regions and plays an important role in specifyinganterior structures (48, 49). Exposed embryos showed a decreaseof anterior shh expression with concomitant microphtalmy andmicrocephaly, as revealed by the reduction of the otx2 domain(Figure 2, compare the space between bars in the control embryoin D with the treated embryo in F). Also, there is a pronouncedshortening of the A-P axis (compare control embryos in Figure2D,E with treated embryos in F,G). In control embryos, thetranscription factor sox9 is expressed in the cranial neural crestcells as they populate the pharyngeal arches, the otic placode,the developing eye, the genital ridges, and also the notochord(Figure 2E) (50). Embryos treated with GBH showed reducedeyes and genital ridges, and developed abnormal pharyngealarches. The migration of neural crest cells to these structureswas delayed, as revealed by a more dorsal position (compareFigure 2G with E).To analyze the effects of glyphosate alone on dorsal midlinedevelopment, we performed bilateral injections at the 2-cellstage. Embryos were fixed when sibling controls reached stage28-30, and the expression of shh was analyzed. To betterunderstand cephalic defects, the pattern of otx2 was alsoexamined. Similar to embryos treated with GBH, we observedreduced prechordal shh expression accompanied by strongmicrocephalic and microphtalmic phenotypes. This is likely dueto a decrease of midline-derived signals (Figure 2H,I). Takentogether, all of these results indicate that GBH as well asglyphosate alone cause cephalic defects that probably result froma reduction of shh and otx2 expression in anterior structures.The delay in the migration of cranial neural crest cells in thetailbud stage embryos together with the inhibition of slugexpression at earlier stages led us to next examine whethercraniofacial development would be impaired in older embryos.GBH and Glyphosate Disrupt the Development of theCraniofacial Skeleton. The pattern of neural crest derivativesin the cranial skeleton of the Xenopus embryo was previouslyestablished (40). Briefly, in the first pharyngeal arch, neural crestcells contribute to the upper (quadrate, Qu) and lower (Meckel’s,Me) jaws; in the second arch, they contribute to the cerathoyalcartilage (Ce), while in the third and fourth arches, neural crestcells contribute to the anterior and posterior regions of thebranchial/gills cartilage (Br), respectively (Figure 3C).To address if the effects seen at neurula and tailbud stagesare correlated with craniofacial malformations, embryos treatedwith GBH and embryos unilaterally or bilaterally injected withglyphosate at the 2-cell stage were allowed to develop up tostage 47 and processed with Alcian Blue staining for skeletalanalysis. The gross morphology of GBH-treated embryosrevealed an overall reduction of cranial structures and mi-crophthalmy (compare Figure 3A,C with B,D). All affectedembryos displayed a reduction of the quadrate and Meckel’scartilages (asterisks, Figure 3D), while the branchial andcerathoyal cartilages were mildly affected.Unilateral glyphosate injections resulted in a general decreaseof Alcian blue staining and in a reduction of the Meckel’s andquadrate cartilages on the injected side (asterisks, Figure 3E,F).In some embryos, the eye practically disappeared from theinjected side (arrow, Figure 3H). Moreover, bilaterally injectedembryos exhibited cyclopia (Figure 3I, arrow), consistent withthe loss of Shh signaling from the prechordal mesodermobserved at earlier stages. Similar results were obtained in frogembryos treated with cyclopamine (Figure 3J), a known inhibitorof the Hedgehog pathway which leads to developmentalmalformations and holoprosencephaly-like abnormalities, in-cluding cyclopia in the most severe cases (51-53). Unilateralinjections of cyclopamine produced cartilage alterations similarto those obtained with glyphosate injections (not shown).In summary, our results are compatible with the malforma-tions observed in the offspring of women chronically exposedFigure 2. GBH and glyphosate produce A-P truncations and impairthe expression of dorsal midline and neural crest markers. (A-I)WMISH analysis at neurula (A-C) and tailbud (D-I) stages. (A)Control embryo hybridized with shh (arrow) and pax6 (white ar-rowheads). (B-C) Embryos exposed to 1/5000 dilution of GBH. Noticethe dramatic reduction of shh expression in the embryonic dorsalmidline (arrows) and the caudal displacement of the anterior limit (greenarrowheads) (85%, n)33). The expression of pax6 is reduced, andthe domain is not properly resolved in the eye field (light bluearrowheads) (85%, n)33). (D,E) Control embryos. (D) Normalexpression of shh in the notochord (n), floor plate (fp), and prechordalmesoderm (pm) and of otx2 in the eye (e), forebrain (fb), and midbrain(mb). The space between bars indicates the size of the brain. (E) Normalexpression of sox9 in the pharyngeal arches (pa), otic placode (op),eye (e), genital ridge (gr), and notochord (n). (F,G) 1/5000 GBH-treatedembryos. (F) Reduced expression of shh and otx2 (92%, n)24) indorsal midline cells (shh, arrow), prechordal mesoderm (shh, blackarrowhead), eye (otx2, yellow arrowhead), and brain structures (otx2,space between bars). (G) Diminution of sox9 expression in thenotochord (black arrow), genital ridge (green arrow), and eyes (yellowarrowhead) (87%, n)30). Notice the delay in the migration of neuralcrest cells toward the pharyngeal arches (red arrowheads). Treatedembryos (F and G) showed microphtalmy, microcephaly (compare thespace between bars in D-G), and a shortened A-P axis (89%, n)54). (H,I) Embryos hybridized with shh and otx2. (H) Control embryoshowing the same structures as those in D. (I) Embryo bilaterallyinjected with 360 pg of glyphosate per cell at the 2-cell stage plusDOG as tracer (green fluorescence in the inset). Similar to that in GBH-treated embryos, shh and otx2 expression was reduced (62%, n)16),and this was accompanied by microcephaly (compare space betweenbars) and microphtalmy (yellow arrowhead).D Chem. Res. Toxicol., Vol. xxx, No. xx, XXXXPaganelli et al.
to GBH during pregnancy (see Discussion). These malforma-tions suggest the loss of midline signaling, accompanied bydefects in neural crest migration (or increased apoptosis) withaberrant development of mandibular and maxillary structures.Phenotype Induced by GBH Is at Least Mediated byChanges in RA Signaling. It was previously reported thatincreasing concentrations of RA caused progressive truncationof anterior and posterior structures in Xenopus laeVis (20, 21).The most severely affected embryos lacked eyes, nasal pits,forebrain, midbrain, and otic vesicles, and displayed truncationsof the tail. The phenotypes produced by GBH and glyphosateresemble the teratogenic effects of embryos treated with RA;therefore, we theorized that the RA pathway could be associatedwith the morphogenetic effects of glyphosate during earlyembryogenesis.The RA signal is transduced through nuclear retinoic acidreceptors (RARs), which control the expression of target genesinvolved in vertebrate pattern formation, organogenesis, andtissue homeostasis (54). Ro 41-5253 (Ro) is an antagonist ofthe RARRreceptor, which is expressed during early develop-ment in Xenopus (16, 17, 55, 56). Ro was previously used as atool to block retinoid-mediated signaling, producing a varietyof morphological changes in the frog embryo. The most severephenotypes showed anterior and posterior truncations, a reduc-tion or loss of eyes and otic vesicles, and a general disorganiza-tion of branchial arches (22). Moreover, maternal insufficiencyof vitamin A (the precursor of RA) or RA in excess invertebrates cause a wide range of teratologic effects (18, 57, 58).All this evidence demonstrates that vertebrates require aprecisely regulated supply of retinoids during embryogenesis.Considering that the phenotypes obtained in our analysispredominantly resemble those of RA excess, we wondered ifGBH treatments are able to increase endogenous RA activity.To answer this question, we measured the levels of RA signalingby taking advantage of the reporter plasmid RAREZ (32), asdescribed in the Experimental Procedures section. Figure 4Ashows that GBH treatment significantly increased the level ofRA signaling in the embryo in a concentration-dependentmanner. Importantly, the RA receptor antagonist Ro rescuedthe effect of GBH since the level of the RA output, as measuredby the reporter assay, was not significantly different from thatin RAREZ-injected, untreated controls (Figure 4A). Together,these observations strongly suggest that GBH increases endog-enous retinoid activity.If an increase of RA signaling underlies the phenotypeproduced by GBH treatments, antagonizing the RA pathwayshould rescue the effect of GBH. To examine this hypothesis,embryos were incubated at the 2-cell stage with GBH alone orwith GBH together with 0.5 or 1 µM Ro added when siblingcontrols reached stage 9 (22). Frog embryos were analyzed bytheir morphological aspect and also were hybridized with shhand otx2 probes.Control embryos showed an expression of otx2 in theforebrain, midbrain, and optic vesicle, while shh transcripts aredistributed along the embryonic dorsal midline (Figure 4B).Embryos treated continuously with GBH showed a down-regulation of shh and otx2, reduced head structures, andshortened A-P axis (Figure 4C). Similar results were obtainedafter treating frog embryos with 0.1 or 1 µM RA (21, 59, 60).As previously reported (22), embryos incubated with 0.5 or 1µM Ro alone also displayed a concentration-dependent shorten-ing of the A-P axis and reduction of head structures, which wasconfirmed by a reduction of the otx2 domain (Figure 4D,E;compare the space between bars with B). We also observed amore diffuse staining of shh, mainly in the prechordal meso-derm, in comparison with that of sibling controls (Figure 4D,E;arrows). When 0.5 or 1 µM Ro was added at stage 9 to embryoscontinuously exposed to 1/5000 dilution of GBH, the elongationof the A-P axis was recovered as well as the normal expressionpattern of otx2 and shh (Figure 4F,G). We conclude that theability of Ro treatment to rescue the teratogenic effect of theGBH supports the idea that RA activity is elevated in GBH-treated embryos.GBH Produces Similar Teratogenic Effects in ChickEmbryos. To test whether the teratogenic effects of GBH arereproducible in an amniote vertebrate, we chose the chick model.Embryos were incubated with 1/3500 or 1/4500 dilutions ofGBH and analyzed at the HH stage 9 (8 somites) by immun-ofluorescence with an anti-Pax6 antibody and by WMISH witha c-shh probe (61). As was previously demonstrated for Pax6mRNA (62), the Pax6 protein is normally distributed in the opticvesicle; in the distinctive comet-like shape in the ectoderm,posterior to the region of the optic vesicle; in the hindbrain inrhombomeres r3 and r5 and along the spinal cord (Figure 5A,D).GBH treatments produced a concentration-dependent reductionFigure 3. GBH treatment and glyphosate injection result in cephalicmalformations and abnormal development of the craniofacial skeleton.(A-D) 1/5000 GBH-treated embryos analyzed at stage 45-47. (A,B)Gross morphology. (A) Control embryo; eyes (arrows); head size (spacebetween yellow bars). (B) Embryo exposed to GBH showing reducedeyes (arrows) and head structures (89%, n)38) (compare the spacebetween yellow bars in A and B). (C,D) Embryos stained with Alcianblue. (C) Control embryo, showing facial cartilages: Meckel (Me),ceratohyal (Ce), infrarrostral (I), quadrato (Qu), and branchial (Br).(D) Reduction of Me and Qu cartilages (asterisks) in GBH-exposedembryos (77%, n)39). (E-I) Embryos injected with 360 pg ofglyphosate per cell in one or both cells at the 2-cell stage and analyzedat stage 47 by Alcian blue staining (E,F) or gross morphology (H,I),which was compared with that of sibling controls (G). (E,F) Unilaterallyinjected embryos showing reduced Alcian blue staining and smallerQu and Me cartilages (asterisks) on the IS (56%, n)16). (G) Controlembryo. Arrows indicate the position of the eyes. (H) Notice thereduction of the eye in the IS (arrow) (54%, n)13). (I) Bilaterallyinjected embryo exhibiting cyclopia (arrow) (38%, n)8). (J)Cyclopamine-treated embryo. Observe the proximity of both eyes(arrows), due to midline defects (compare with the control embryo inG). IS, injected side. NIS, noninjected side. Gly-inj, embryo injectedwith glyphosate.Teratogenic Effects of GlyphosateChem. Res. Toxicol., Vol. xxx, No. xx, XXXX E
of the optic vesicles, as revealed by a reduction of thecorresponding Pax6 domain, and this was accompanied bymicrocephaly (compare the space between bars in Figure 5B,Cwith A). We also observed a gradual loss of the r3 and r5domains in embryos treated with GBH (compare Figure 5E,Fwith D), which resembles the results observed in frog embryosin the krox-20 domains (Figures 1B and 2E). Hybridization withthe c-shh probe showed that, as in Xenopus, the prechordalmesoderm domain is preferentially lost in GBH-treated chickembryos (compare Figure 5G with H,I). As the GBH concentra-tion increases, the expression along the embryonic dorsal midlinealso gradually disappears (Figure 5H,I).Therefore, our experiments with chick embryos further extendconclusions from studies about the teratogenic effects of GBHin amphibians to other vertebrate species.DiscussionThe results presented above argue that both GBH andglyphosate itself interfere with key molecular mechanismsregulating early development in both Xenopus and chickenembryos, leading to congenital malformations. Sublethal dosesof the herbicide (430 µM of glyphosate in 1/5000 dilutions ofGBH) and injections leading to a final concentration of 8 to 12µM of glyphosate in the injected side of the embryo weresufficient to induce serious disturbances in the expression ofslug, otx2, and shh. These molecular phenotypes were correlatedwith a disruption of developmental mechanisms involving theneural crest, embryonic dorsal midline formation, and cephalicpatterning. Because glyphosate penetration through the cellmembrane requires facilitation by adjuvants present in com-mercial formulations (5, 6), we tested the effects of glyphosatealone by directly microinjecting it into Xenopus embryos. Thesimilarity of the phenotypes obtained in both situations suggeststhat they are attributable to the active principle of GBH andnot to the adjuvants.We will discuss our results in the following context: (1) thecorrelation of our phenotypes with those observed in animalmodels with an impairment of RA signaling or deficits in theexpression of critical genes that control embryonic development;(2) the probable mechanisms underlying the phenotypes inducedby GBH and glyphosate; (3) possible correlations with clinicalcases of human offspring exhibiting malformations in zonesexposed to GBH.Misregulation of RA, shh, and otx2 Are Involved inCephalic Malformations and Neural Crest-Derived Pheno-types Reminiscent of the Effects of GBH and Glyphosate.The phenotypes obtained after GBH treatments or injections ofglyphosate alone are strikingly reminiscent of those observedas a consequence of an excess of RA signaling in vertebratesand humans. Acute or chronic increase of RA levels leads toteratogenic effects during human pregnancy and in experimentalFigure 4. Phenotype induced by GBH is mediated by an increase ofRA signaling (A). Analysis of RA activity with the reporter plasmidRAREZ. All embryos were injected with the reporter plasmid RAREZ,except for uninjected controls, and left untreated or were treated asindicated in the figure until stage 14-15, when they were processed.Results are expressed as arbitrary luminiscence units per µg of protein.A two-tailed t test was employed to analyze the significance in thedifference of the means. ** p < 0.01; *** p < 0.0001. (B-G) WMISHfor shh and otx2 at tailbud stages. (B) Control embryo. Notochord (n);floor plate (fp); brain (space between bars), eye (arrowhead). (C)Embryo treated with 1/5000 GBH manifesting microcephaly (spacebetween bars), reduced eyes (arrowhead), diminished Shh signalingfrom the prechordal mesoderm (arrow), and shortened A-P axis (78%,n)9). (D,E) Embryos incubated with 0.5 and 1 µM RA antagonistRo 41-5253, displaying a reduction in the otx2 domain accompaniedby microcephaly (bars) and microphtalmy (arrowhead), and morediffuse expression of shh (arrows) (80%, n)15 for 0.5 µM Ro; 87%,n)15 for 1 µM Ro). (F,G) Embryos treated with 1/5000 GBH at the2-cell stage; 0.5 µM Ro (F) or 1 µM Ro (G) was added at stage 9, andphenotypes were analyzed at the tailbud stage. Notice that Ro revertsthe phenotype produced by GBH, rescuing the A-P axis elongationand the expression of shh and otx2 (compare with the control embryoin B) (88%, n)17 for 0.5 µM Ro, which gives the best rescue effectsince the effect of the retinoid antagonist begins to prevail with 1 µMRo). All embryos are oriented with the anterior end toward the right.Figure 5. Teratogenic effects of GBH in chicken embryos. (A-C)Whole-mount inmunofluorescence analysis of Pax6 at 8 somites. (A,D)Control embryo showing Pax6 expression in the optic vesicles(arrowheads in A) and in rhombomeres r3 and r5 (blue arrows in D).(B,E and C,F) graded reduction of Pax6 expression in embryos treatedwith 1/4500 and 1/3500 dilutions of GBH, respectively. Notice theprogressive microcephaly (compare space between bars with D) andthe loss of Pax6 expression corresponding to rhombomeres r3 and r5(red arrows). The remaining fluorescence corresponds to specific Pax6expression that is normally found in the spinal cord but is out of focusin the control embryo in D. (G-I) WMISH with c-shh. (G) Controlembryo. Shh transcripts are seen in dorsal midline cells (black arrow)and in the prechordal mesoderm (green arrow). (H,I) Embryos treatedwith 1/4500 and 1/3500 dilutions of GBH, respectively. Notice theabolishment of shh expression in the prechordal mesoderm (dotted greenarrow) and the progressive decrease of shh expression in the midlinecells in a concentration-dependent manner (dotted black arrows).F Chem. Res. Toxicol., Vol. xxx, No. xx, XXXXPaganelli et al.
models. The characteristic features displayed by RA embry-opathy in humans include brain abnormalities such us micro-cephaly, microphthalmia, and impairment of hindbrain devel-opment; abnormal external and middle ears (microtia or anotia);mandibular and midfacial underdevelopment; and cleft palate.Many craniofacial malformations can be attributed to defectsin cranial neural crest cells (19, 24).This spectrum is consistent with the phenotypes obtained inrodent models exposed to RA. When administered duringgastrulation in mice, RA severely impairs the development ofthe anterior neural plate, resulting in ocular, brain, and facialmalformations. Exposure at critical stages of neural crest cellmigration induces craniofacial malformations comparable tothose seen in Di-George syndrome. Later exposure, when theepibranchial placodes are active, results in mandibulofacialdysostosis-like syndromes (19). These authors suggest thatexcessive cell death in regions where apoptosis normally takesplace may underlie a general mechanism for craniofacialmalformations associated with teratogens.An excess of RA signaling is able to down-regulate shhexpression in the embryonic dorsal midline in Xenopus (60, 63).Shh deficiency is associated with holoprosencephaly syndrome(HPE), a CNS malformation with a frequency of 1/250 ofpregnancies and 1/10000 of live births. HPE is a defectgenerated by the deficiency of the embryonic dorsal midline,leading to a failure in the division of the brain hemispheres.This results in unilobar brain, cyclopia, and defects in the closureof the dorsal neural tube, accompanied by other defects includingmicrocephaly, abnormally decreased distance between the eyes(hypotelorism), proboscis, and cebocephaly (a simple nose)(51-53). Moreover, Shh signaling is necessary for the develop-ment of the cranial neural crest derivatives. In the mouse,specific removal of Shh responsiveness in the neural crest cellsthat give rise to skeleton and connective tissue in the headincreases apoptosis and decreases proliferation in the branchialarches, leading to facial truncations (64). In zebrafish, the cranialneural crest requires Shh signaling emanating from the embry-onic dorsal midline and the oral ectoderm to achieve correctmigration and chondrogenesis (65). In chicken embryos, de-velopment of the lower jaw skeleton requires Shh signaling fromthe foregut endoderm to prevent apoptosis of the neural crestcells that migrate to the first branchial arch (66). Shh signalingfrom the ventral midline is necessary, as an antiapoptotic agent,for the survival of the neural epithelium, and it is also essentialfor the rapid and extensive expansion of the early vesicles ofthe developing midbrain and forebrain (67-69).An excess of RA signaling also down-regulates otx2 expres-sion in Xenopus, chicken, and mouse embryos (24). Knockoutmice for otx2 lack all the brain structures anterior to rhombomere3. Interestingly, heterozygous mutants showed craniofacialmalformations including the loss of the eyes and lower jaw(agnathia). These phenotypes are reminiscent of otocephalyreported in humans and other animals and suggest that otx2 playsan essential role in the development of cranial skeletons ofmesencephalic neural crest origin (70-72).Otx2, in turn, is necessary for the expression of shh in theventral midbrain (73). All this evidence indicates that RAsignaling, otx2, and shh are part of a genetic cascade criticalfor the development of the brain and craniofacial skeleton ofneural crest origin. Glyphosate inhibits the anterior expressionof shh, reduces the domain of otx2, prevents the subdivision ofthe eye field, and impairs craniofacial development, resemblingaspects of the holoprensecephalic and otocephalic syndromes.This prompted us to investigate whether an increase of RAsignaling could be mediating the effects of GBH treatments.GBH Increases the Activity of the Morphogen RA,Leading to Teratogenic Effects. In Xenopus embryos, theendogenous activity of retinoids gradually increases during earlyembryogenesis and is finely regulated in space. At late gastrula,a rostral-caudal gradient from 0.01 to 0.16 µM RA isestablished, with the highest levels at the posterior end of theembryo. The gradient persists at the early neurula stage (stage13-14). Synthesis and degradation of RA seem to be themechanisms that lead to this uneven distribution (74). Thisgradient explains why low doses of applied RA primarily affectthe cephalic region and increasing the doses begins to affectthe trunk (20, 21). Moreover, maintaining a normal endogenousdistribution of RA is important for axes patterning and orga-nogenesis not only in Xenopus (74, 22, 38) but also in otherVertebrates such as zebrafish (75-77), chicken (78-80), andmouse embryos (81).In this study, GBH treatments or glyphosate injections mostlyreproduce the morphological phenotype obtained after treatmentsof Xenopus embryos with RA concentrations from 0.1 to 10µM (21). The fact that GBH treatments increase endogenousRA activity, as measured by the RAREZ reporter, and that theGBH-induced phenotypes are rescued by the antiretinoid Rostrongly suggest that augmented RA activity is a major causeof the molecular and morphological phenotypes described inthis work.GBHs are considered endocrine disruptors because of theirability to impair the synthesis of steroid hormones (82).Glyphosate inhibits the activity of aromatase, a member of thecytochrome P450 family crucial for sex steroid hormonesynthesis (4). Retinoid activity is regulated by degradation ofRA by the CYP26 enzymes, which are members of thecytochrome P450 family and are present in all vertebrates fromearly stages of embryogenesis. Transcription of CYP26 isdevelopmentally and spatially regulated. Deficiencies of thisenzyme produce serious malformations in different vertebratemodels consistent with an important increase in RA signaling.These phenotypes include cephalic defects, abnormalities of theeye and the forebrain, agnathia, and caudal truncations (83-90).In this context, it will be interesting to elucidate in the future ifthe increase of RA signaling induced by GBH could be aconsequence of inhibiting the activity of CYP26 enzymesresponsible for maintaining a normal RA distribution by specificterritorial degradation.In Xenopus laeVis, RA favors the differentiation of primaryneurons (39, 60, 91). Since GBH increases retinoid signaling,the reduction in the number of primary neurons in GBH-treatedand glyphosate-injected embryos is paradoxical. Other bio-chemical mechanisms could be triggering the inhibition ofneurogenesis. For example, we cannot rule out that apoptosisof neural precursors could be involved in this process since GBHand glyphosate have a toxic effect on mitochondrial membranesand activate caspases3/7 (7). Both GBH and glyphosate inhibitshh expression, and the Shh protein is known to have anantiapoptotic function, necessary for the survival of the neu-roepithelium (67, 68). Abnormal induction of cell death is oneof the crucial mechanisms of malformations associated withdifferent teratogenic agents such as ethanol, RA, hypoxia, andchemicals herbicides (19, 92).Assuming a linear response of the luminescence system withthe RAREZ reporter used to measure RA signaling, we estimatethat the endogenous concentration of RA available for activityin Xenopus embryos is around 0.2 µM (Figure 4A, compareTeratogenic Effects of GlyphosateChem. Res. Toxicol., Vol. xxx, No. xx, XXXX G
RA bars with the RAREZ bar). This is very similar to theaverage concentration of 0.15 µM previously measured byHPLC (20). Importantly, treatments with 1/5000 dilution ofGBH do not show a significant increase of RA activity whencompared to that of untreated controls, as measured by thereporter system (Figure 4A). However, this dilution clearlyproduces cephalic and trunk phenotypes and craniofacialmalformations, as shown throughout this work, which arerescued by Ro treatments. Therefore, the RAREZ reporter doesnot seem to be sensitive enough to detect minimal variations inthe levels of RA activity. This reinforces the importance of usingvertebrate embryos as biosensors for testing possible teratogens.Moreover, it has been recently reported that Triadimefon, asystemic fungicide with teratogenic effects in rodent models,produces craniofacial malformations in Xenopus laeVis byaltering endogenous RA signaling (93). Arsenic, anotherendocrine disruptor, also increases RA signaling at low,noncytotoxic doses, in human embryonic NT2 cells (94). RAsignaling is one of the finest pathways to tune up gene regulationduring development, and all this evidence raises the possibilitythat disturbances in RA distribution may be a more generalmechanism underlying the teratogenic effects of xenobiotics invertebrates. Since mechanisms of development are highlyconserved in evolution among vertebrates (95), we would liketo stress that they could be useful as very sensitive biosensorsto detect the undesirable effects of new molecules.Clinical Approaches. In Argentina, the extension of soildevoted to transgenic soy reached 19 million hectares. Twohundred million liters of glyphosate-based herbicide is used fora production of 50 million tons of soy beans per year (96, 97).The intensive and extensive agricultural models based on theGMO technological package are currently applied withoutcritical evaluation, rigorous regulations, and adequate informa-tion about the impact of sublethal doses on human health andthe environment, leading to a conflicting situation. In this work,we focused on sublethal doses of GBH to arrive at the thresholdsfor teratogenic phenotypes instead of lethality.In the last 10 years, several countries in Latin America haveinitiated studies about the environmental consequences of theuse of herbicides and pesticides. In Paraguay, an epidemiologicalstudy in the offspring of women exposed during pregnancy toherbicides showed 52 cases of malformations (3), whichstrikingly resemble the wide spectrum phenotypes resulting froma dysfunctional RA or Shh signaling pathway. In Argentina, anincrease in the incidence of congenital malformations began tobe reported in the last few years (Dr. Hugo Lucero, UniversidadNacional del Nordeste, Chaco; personal communication). InCórdoba, several cases of malformations together with repeatedspontaneous abortions were detected in the village of Ituzaingó,which is surrounded by GMO-based agriculture. These findingswere concentrated in families living a few meters from wherethe herbicides are regularly sprayed. All of this information isextremely worrying because the risk of environmentally-induceddisruptions in human development is highest during the criticalperiod of gestation (2 to 8 weeks) (98). Moreover, the maturehuman placenta has been shown to be permeable to glyphosate.After 2.5 h of perfusion, 15% of administered glyphosate istransferred to the fetal compartment (99).All of the evidence reported in the scientific literature andthe clinical observations in the field were not sufficient, however,to activate the precautionary principle of the environmentallegislation in order to realize the depth of the impact on humanhealth produced by herbicides in GMO-based agriculture. Toour knowledge, the results presented in this work show for thefirst time that at least some of the malformations produced byGBH in vertebrate embryos are due to an increase of endogenousRA activity, consistent with the well-known syndrome producedby an excess of RA.Acknowledgment. We acknowledge the following research-ers for providing us with the constructs for making probes:David Wilkinson for krox-20, Michael Sargent for slug, NancyPapalopulu for N-tubulin, Ira Blitz for otx2, Jean-Pierre SaintJeannet for sox9, Thomas Hollemann for pax6, and Cliff Tabinfor c-shh. We are also grateful to Abraham Fainsod for theRAREZ plasmid, Dr. M. Klaus for providing Ro 41-5253, andBruce Blumberg for useful discussions. We thank Ana Adamofor material support, Hugo Rıó Ezequiel Varela, and ErnestoGonzález for helping us with chicken experiments, and membersof our lab (Cecilia Aguirre, Sabrina Murgan, and DiegoRevinski) for helping with embryos and reagent preparations.We also thank Carlos Davio and Sandra Verstraeten forassistance in luminiscence determination. A.E.C. is particularlyindebted to Bar de Cao. A.R.P. and A.E.C. are from ConsejoNacional de Investigaciones Cientı´ficas y Técnicas (CONICET)and Universidad de Buenos Aires. V.G. was supported by afellowship from ANPCyT, and H.A was supported by afellowship from Universidad de Buenos Aires. S.L.L. is fromCONICET. This work and the authors are completely indepen-dent from industry. The authors declare no competing financialand commercial interests.References(1) vom Saal, F., and Hughes, C. (2005) An extensive new literatureconcerning low-dose effects of bisphenol A shows the need for a newrisk assessment. EnViron. Health Perspect. 113, 926–933.(2) Myers, J., Zoeller, R., and vom Saal, F. (2009) A clash of old andnew scientific concepts in toxicity, with important implications forpublic health. EnViron. Health Perspect. 117, 1652–1655.(3) Benıt Leite, S., Macchi, M. A., and Acosta, M. (2009) Malforma-ciones Congénitas asociadas a agrotóxicos. Arch. Pediatr. Drug 80,237–247.(4) Richard, S., Moslemi, S., Sipahutar, H., Benachour, N., and Seralini,G. E. (2005) Differential effects of glyphosate and roundup on humanplacental cells and aromatase. 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