[Frontiers in Bioscience E2, 133-142, January 1, 2010]
Margherita Napoletano1, Davide Pennino1, Gaia Izzo2, Salvatore de Maria2, Raffaele Ottaviano1, Maddalena Ricciardi1, Roberto Mancini1, Antonella Schiattarella5, Ernesto Farina4, Salvatore Metafora1, Maria Cartenì2, Alberto Ritieni3, Sergio Minucci2, Franco Morelli1
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TABLE OF CONTENTS
1. ABSTRACT
Ochratoxin A (OTA) is a mycotoxin produced by fungal of Aspergillus species absorbed in human through contaminate food in gastrointestinal tract. OTA has been demonstrated to be teratogenic in a number of species including mice and potentially human. Mice exposed in uterus to OTA develop craniofacial abnormalities such as exencephaly, microencephaly, microphthalmia and facial clefts. An important role in differentiation of maxillofacial are exerted by the Hox related genes Dlx and Msx. In the present investigation we have confirmed that 2.75 mg/kg body weight OTA, given at gestational day 7.5, induces significant developmental craniofacial anomalies in mice and we have demonstrated the down expression of Dlx5, a member of Dlx gene family, that seems to be responsible of the observed deformities. These results support the hypothesis that Dlx5 is a target for ochratoxin and the inhibition of its function, directly or indirectly, could be at origin of the observed differentiation defects.
2. INTRODUCTION
Ochratoxin A (OTA) is a nephrotoxic, carcinogenic and teratogenic mycotoxin produced as a secondary metabolite by certain fungal of Aspergillus and Penicillium species (1). Mycotoxins as ochratoxin A, Citrinin and Sterigmatocystin, are isocumarinic derivatives of phenylalanine and are widespread contaminants of grains and agricultural products. OTA is commonly found in animal feeds and human foodstuffs including cereals products, dried fruit, dried fish, coffee (2;3). Considerable levels of OTA have also been found in red wines at concentrations up to 7 mg/l, meat and meat products, and in confectionary with contamination levels ranging from 0.1-3.8 mg/ kg (4) (3). In particular, the fungal production of this mycotoxins is optimized by humidity ranging 15-19% and temperature more than 15�C and pH 5.5 (5).
The OTA is absorbed in human through contaminate food in gastrointestinal tract, entheropatic circulation and proximal tubule of nephron (6) accumulating in kidney, moreover is vehiculated and stabilized in serum by albumin, accumulating in lung, liver, kidney, hearth, adipose tissue and gut at different concentrations. Individual variation in plasma levels of ochratoxin A are founded in humans. (3).
Exposure to OTA has been demonstrated to be teratogenic in a number of species including rats (7), mice (8), hamsters (9), chick (10) and potentially humans.
Surprising despite the fact that OTA is a specific and potent animal teratogen, largely distributed in food and detected in all human plasma samples (11) with an half-life in humans approximately 6-13-fold longer than that reported in rats (12, 13) and that children and infants have been suggested to be more at risk from the toxic effects of OTA, very little or no research has been undertaken to identify possible risks for human fetal development.
Many effects of OTA administration in vivo in rats and mice are also observed in humans, but it is not known the mechanism.
The most common defects observed in mice exposed in uterus to OTA are craniofacial abnormalities such as exencephaly, microencephaly, microphthalmia, facial clefts, and hypoplastic jaws, all of which appear to be dependent on both the dose and the period of gestation during which the toxin is administered (13).
The period of gestation in which there is, in mice and in rat, the major sensibility to OTA, is between the day 6 and 8 post coitum (pc) (14, 15) with a minimal dose of 3mg/Kg body weight.
The timing of these effects coincides with the period of neurulation. Once the neuropores are closed, subsequent exposure has not been observed to be teratogenic. This may be due, probably, to reduced placental transfer later in development (16, 17) and for a specific action of the toxin on the developing nervous system (18, 19, 20).
For this reason it seems interesting to make an analysis of embryological malformations induced in mice by OTA exposure and evaluate the genetic events that are involved in these mechanisms. Our interest was to understand what genes are target for OTA, with particular interest for the genes involved in differentiation and, in particular, in the differentiation of maxillary and craniofacial. We focused our attention on homeotic genes and on some homeotic related genes.
For the correct morphogenesis of the different segments of mouse embryo are necessary the concerted action of Hox genes (21). These genes constitute a highly conserved family of homeobox genes that act as transcription factors. An important role in differentiation of maxillofacial are exerted by Dlx, Msx and Otx genes however, their mode of action as regulatory molecules, might be more complex as it has been shown that members of the Dlx family can form dimeric complexes with Msx homeoproteins mutually affecting their DNA-binding properties (22). With respect to their biochemical property Msx proteins act as transcriptional repressor, while Dlx protein are transcriptional activator (23).
In the mouse, there are at least six Dlx genes arranged as pairs and located near Hox clusters (Dlx1 and Dlx2 near HoxD; Dlx3 and Dlx7 near HoxB; Dlx5 and Dlx 6 near HoxC) (24, 25).
Dlx genes are all expressed in spatially and temporally restricted patterns in craniofacial primordia, basal telencephalon and diencephalon. The pattern of expression of Dlx5 differs from that of the other members of the family, in fact Dlx5 is expressed much earlier than other Dlx genes (26) during development in territories that define the rostral and lateral border of the neural plate and the rostral prosencephalon, moreover Dlx5 and Dlx6 are expressed in all developing bones from the time of initial cartilage formation (24, 27).
A further indication of the possible importance of Dlx5 in the control of bone differentiation comes from a study (28) in which it has been shown, that this gene is expressed at specific stages of osteoblast differentiation and could repress the osteocalcin gene expression by interacting with a single homeodomain-binding site in its promoter (29).
Studies of targeted inactivation of Dlx 5 genes, as reported by Acampora et al. (29) , shown as homozygous mutant not survive longer than 24 hours after birth, and about the 12% of embryos presents exencephaly in addition to other severe phenotype modifications. Dlx5 expression in brain begins around 10 days post coitum (dpc) and the first phenotypic difference, induced by the Dlx5 homologous recombination inactivation, appears at 12.5 dpc, while craniofacial malformations were well visible at 14.5 dpc. Mutant embryos could be recognized by their shorter snout and open fontanelle, while in the few exencephalic embryos the craniofacial defects were more conspicuous. Moreover Dlx5 mutant embryos at 14.5 dpc present severe malformations in maxillary region with a cleft secondary palate and the loss of horizontal laminae of the palatine bones, moreover the nasal and maxillary bones are shorter, resulting in a general reduction of the length of the snout. The palatine processes of the maxilla are reduced especially with respect to their posterior development and they fail to form proper connections with the palatine bones (29) .
At E16.5 Dlx5 mutant have defects in olfactory placode and hypoplasia of frontonasal prominence derivatives more in particular they present pronounced asymmetry of nasal capsule with a nearly complete loss of the right nasal apparatus (30).
On the bases of these considerations we wanted to evaluate the possible regulatory effect, induced by OTA administration during a critical moment of gestation, on expression of some homeotic related genes in order to understand the possible mechanism of the mycotoxin in the induction of differentiation defects. We have concentrated our attention principally on the two homeotic gene Dlx5 and Msx1, that have opposite roles in the regulation of transcription, in fact, as mentioned above, Msx proteins act as transcriptional repressors, while Dlx protein are transcriptional activators (23, 31).
3. MATERIALS AND METHODS
3.1. Experimental animals
Sexually mature C57Bl6 female mice (Charles River Laboratory) were maintained on standard conditions, feed and water available ad libitum, in a temperature controlled and artificially illuminated room (12-h light:12-h dark cycle), free from any source of chemical contamination. After an acclimatization period of 1 week, females were mated with mature males of the same strain and the day on which were found vaginal plug was designated as day zero of pregnancy. After mating, the female mice were individually housed in polypropylene cages.
3.2 Ochratoxin Treatments
Pure ochratoxin A (Sigma Chemical Ltd), was dissolved in 0.1 M Sodium Bicarbonate solution. Pregnant female were injected intraperitoneally (i.p.) with a single dose of OTA at 3.0 mg/Kg body weight, in 100�l of 0.1M Bicarbonate vehicle, on day 7.5 of gestation and embryo were taken at day 16 (E16) post coitum (pc). Control mice was injected at same time with only 100�l of sodium bicarbonate vehicle.
3.3. Embryo analysis
Pregnant, OTA treated, females and control animals were killed by cervical dislocation on gestation day 16. Embryo E16 were removed from uterus and fixed in Bouin's fixative (1% saturated picric acid, 5% acetic acid, 24% formaldehyde, in distilled water).
The fixed embryo were photographed to study the morphology and the differentiation defects induced by OTA treatments. After no more than three days the embryo were washed in 70% ethanol, embedded in paraffin, according standard protocols, and sectioned at 5 �m slide for In situ Hybridization (ISH).
3.4. Dlx5 and Msx1 gene fragments purification
A 216 bp Dlx5 and 350 bp Msx1 gene fragments were prepared by reverse transcription polynucleotide chain reaction (RT-PCR) starting from total mRNA extracted from E15 mouse embryo.
Total mRNA was extracted from E15 embryo brain according Chomczynski and Sacchi method, using RNAzol (Invitrogen Co. Ltd) according the manufacturer's instructions and the integrity of purified RNA was verified by agarose gel electrophoresis. For reverse transcription 2 �g of total RNA in a final volume of 20 �l was reverse-transcribed by Avian myeloblastosis virus (AMV) reverse transcriptase (Gibco BRL- Invitrogen Ltd) according manufacturer's instruction in presence of random examer primers (Promega Ltd) at 37�C per 60 min.
PCR amplification of Dlx5 genes fragment (216 bp), Msx1 (350 bp) and Actin (500 bp) was performed by using a Gene Amp PCR system 9700 (Applied Biosystem Ltd) and hot start Taq Gold (Applera Ltd). Actin was used as housekeeping control gene. The Dlx5, Msx1 and Actin PCR primer were: Dlx5 Fw cca gcc aga gaa aga agt gg; Dlx5 Rw tca cc gtg ttt gcg tca gt; Msx1 Fw agc tct gct gcc cta tac ca; Msx1 Rw ggg ctc atc tct tga agc ac; Actin Fw gac tac ctc atg aag atc ct; Actin Rw gct tgc tga tcc aca tct gc. The PCR condition was for Dlx5 and Msx1: initial denaturation at 95� for 10' followed by 36 cycles: 95�, 45''; 53� , 45'' and 72� 45'' with a final extention at 72� for 10', while for Actin initial denaturation at 95� for 10' followed by 32 cycles: 95�, 45''; 60�, 45'' and 72� 45'' and with a final extention at 72� for 10'. The amplification products were run on 1% agarose gel electrophoresis in 0.5 x TBE (Tris Borate EDTA) buffer for the control of the amplicons length.
3.5. Dlx5 and Msx1 riboprobe preparation for ISH
For the preparation of Dlx5 and Msx1 DIG-labelled riboprobes, the Dlx5 216 bp and the Msx1 350bp fragments, obtained by PCR, were purified from agarose gel by electroeluition and inserted in the PCR cloning vector pGEM T easy (Promega Co. Ltd) according the manufacturer's instructions, to obtain the pGEM-Dlx5 and pGEM-Msx1 recombinant plasmids.
The cloned Dlx5 and Msx1 exact sequences and the fragments orientation was controlled by sequencing using an automatic system (Primm Sequencing Core; Primm Italy) that shows how these fragments were both oriented in direction 3'-5'within the recombinant plasmids.
For the sense and anti-sense RNA-probes synthesis, 4�g of pGEM-Dlx5 and pGEM-Msx1 recombinant plasmids were linearized respectively with the restriction enzyme Nco I for the riboprobe sense (Sp6 transcription) and Sal I for the riboprobe anti-sense (T7 transcription), obviously, Nco I and Sal I restriction site are not present in the Dlx5 and Msx1 cloned sequences.
For the synthesis of sense and anti-sense DIG labelled RNA-probes, 1 �g of each linearized plasmid was transcribed using a DIG RNA labelling mix from Roche (Roche Applied Sciences Germany) according the manufacturer's instructions and the Dlx5 and Msx1 riboprobes, purified by ethanol precipitation in presence of 4 M Lithium chloride, were quantified by electrophoresis on agarose gel.
3.6. Dlx5 and Msx1 in situ hybridization
Five-micrometer paraffin sections were dewaxed in xilol twice for 5' each time, rehydrated in graded concentrations of ethanol and rinsed in diethilpyrocarbonate-treated PBS. The sections were fixed in 4% paraformaldehyde in 0.5M NaCl, 0.1M MOPS, pH 7.5, for 30' at room temperature, then washed in 1x PBS. The slides were treated with 10μg/ml protease K in 100 mM Tris-HCl and 1mM EDTA at pH 7.2 for 7' at room temperature and rinsed in 1x PBS for 5' and then transferred in 5x SSC twice for 2' each time. After pre-hybridation performed at room temperature for 30' in Tris-glycine buffer at pH 7.2, the sections were hybridized, in a humidified chamber, overnight at 60�C in a buffer containing 40% deionized formamide, 5x SSC, 1x Denhardt's solution, 100 �g/ml sonicated salmon testes DNA, 100 �g/ml transfer RNA and 80 ng digoxigenin-labeled Dlx5 or Msx1 complementary RNA probe. After incubation, the slides were washed 3 times in 5x SSC for 20' each and then in posthybridization buffer (0.5x SSC, 20% deionized formamide) at 60�C for 40 ' and incubated in NTE (0.5M NaCl, 10 mM Tris-HCl, 5 mM EDTA, pH 7.0) containing 10 �g/ml ribonuclease A for 30' a 37�C.
The slides were rinsed in NTE for 15' at 37�C, then washed in post-hybridization buffer for 30 min at 60�C and rinsed in 2x SSC for 30' at room temperature. The sections were incubated in 1% blocking solution (1% blocking reagent Roche Diagnostics, Basel, Switzerland) in MBT buffer, (0.1M maleic acid, 0.15M NaCl, pH 7.5) for 10' before the overnight incubation at 4�C with an alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (Roche Diagnostics), diluted 1:2000 in MBT buffer. The slides were rinsed 4 times in TBS (25 mM Tris-HCl pH 7.5, 0.15M NaCl, 2.5mM KCl) pH 7.4 for 10' each and then in solution B (0.1% Tween 20, 0.5 mg/ml levamisol) for 10'. The colour detection substrate: 1ml BM purple, 10 �l of 100x solution B (Roche Diagnostics), was applied and the incubation was carried out overnight in the dark at room temperature. The reaction was terminated by rinsing the sections in PBS 1x, 1mM EDTA for 10' at room temperature. The slides were dehydrated and mounted.
4. RESULTS
4.1. Differentiation defects induced by ochratoxin A treatment
The OTA treatment at a very early gestation time consists in a very dramatic differentiation defect, specially concentrated in maxillary craniofacial body segment.
When we compare the normal head development, in untreated mice (Figure 1A), with the pups derived from OTA treated dam we may observe, in the same progeny, different malformation degree not apparently correlated to an experimental difference in the procedure or in quantity and timing of drug administration. On the basis of these observation we can confirm a generalized high toxicity with a different sensibility of different embryos ranging from little to monstrous malformation. In fact, in the same progeny, we observe embryos with not apparently severe differentiative defect, consisting essentially in loss of symmetry in maxillo-facial formation (Figure 1 B) or a more severe deformity with a loss of an ocular formation with replacement of a large central eye (Figure 1 C). On the other hand we can observe progeny with a very high malformation degree probably correlated to a major dam sensitivity to the drug. In fact, embryo derived from an other mouse present in our experiment very dramatic generalized malformation with macro encephalocele in frontal region (Figure 1 D) or monstrous loss in head formation as in (Figure 1 E) or a severe exencephaly with absence of cranial formation (Figure 1 F).
4.2. Dlx5 in situ hybridization
In E16 embryos obtained from pregnant mice that did not received OTA we observed high Dlx5 gene expression in almost all brain and maxillo-facial structures (Figure 2 A), which were morphologically highlighted by the expression of Dlx5 so that we could detect easily the differences in malformations induced by OTA treatment.
Interestingly Dlx5 expression was very reduced in almost all embryonic structures when we administered OTA at pregnancy day 7.5. More in detail, in the different craniofacial malformed structures we found an altered Dlx5 expression pattern. In particular, in OTA treated deformed mice, we observed an absence of correct development of the facial prominence together with absence of the face bone, olfactory epithelium and palatal-sheet development, of course in these structures there was a confused or absent expression of Dlx5, that was instead clearly expressed in the same parts in control mice (Figure 2 B, C).
The wisker-follicles, that clearly identify rostral prominence and olfactory part and show a strong Dlx5 expression in control mice (Figure 2 A), are not evident in OTA treated embryos, in agreement with a strong anatomical alteration in the development of this body district.
Any Dlx5 expression was found in tongue and in the face bone, because these structures was strongly reduced in size and deformed (Figure 2 B,C).
Some embryos, obtained from OTA treated mothers, as results from morphological analysis (Figure 1 C), seem to be monocular showing just one big pseudo eye in the middle of the face and they presented also a Dlx5 expression overlapping between retina and the hypothalamus inferior part, that seems to correspond to an anatomical overlapping of these structures, quite confused in treated embryos (Figure 2 B). At contrary in the normal mice the retina and hypothalamus Dlx5 expression was so clear to identify the anatomical boundary of the structures (Figure 2 A).
Despite a quite normal developed cranial box, analyzing the brain tissues, we found an evident down expression of Dlx5 in all the brain structures, while in normal mice there was a clear and strong expression of the gene in neocortex, striatum, thalamus and hypothalamus, superior and inferior colliculus, tegmentum, inner and middle ear (Figure 2A). Much more difference of Dlx5 hybridization, between treated and untreated mice, was observed in pons, medulla and cerebellum (Figure 2 C) that were considerably damaged by OTA administration.
It is important to underlie that, in OTA treated mice, the absence of Dlx5 expression in some anatomical parts is due to the absence of the correct structures formation.
4.3. Msx1 in situ hybridization
The results obtained from Dlx5 In situ Hybridization induce us to hypothesize the existence of a negative regulation of genes involved in the craniofacial differentiation after treatment with ochratoxin A.
On this base also the Msx1 gene, that acts as transcriptional inhibitor, contrary to Dlx5, could be hyper expressed or remain constant in a system were we hypothesize the inhibition of a genetic pattern.
The Msx1 In situ Hybridization results have not demonstrated differences in the expression rate of this gene and considering that Msx1 is very little expressed in embryo mouse at stage E14 (23) and E16, and that the expression of this gene does not increase in embryo derived from OTA treated mice (Figure 3 A, B) we can arrive to the conclusion that OTA do not interact with Msx1 promoter and that there is not inhibition induced by a major presence of Msx1 gene product.
More in deep we could hypothesize that genes controlled by Msx1 are not down regulated from a major presence or msx1 protein, after OTA treatment.
The teratogenic effects observed, after OTA administration, are probably correlated to the loss of transcriptional activation of genes controlled from the Dlx5 gene product.
5. DISCUSSION
The present study, was undertaken to determine as a single intra peritoneal dose of ochratoxin A (2.75 mg OTA/kg body weight), was teratogenic for the offsprings of pregnant C57Bl6 mice, when given at the 7.5 gestation day, during the major organogenesis period, and if there is a subset of gene, target of OTA, that are responsible for the correct development of a particular body segment, in the aim to explain what is the possible mechanism for the induction of differentiation defects observed after administration of ochratoxin A or others mycotoxins.
OTA has been suggested by various researchers to mediate its toxic effects via induction of apoptosis, disruption of mitochondrial respiration and/or the cytoskeleton or via generation of DNA adducts (32) .
Teratogenic effects of OTA have been well documented in mice (33), with craniofacial abnormalities being the most commonly observed malformations (14).
High lipophilic nature, efficient absorption of OTA from the gastrointestinal tract, an extremely high affinity to the serum albumin and other macromolecules, with the consequent extremely long serum half-life (34), and a very low extent of biotransformation led to persistence of OTA in the body of consumers (35) .
In the present investigation we have confirmed that 2.75 mg/kg body weight OTA proved to be an effective dose to exert significant developmental toxicity in the foetuses of the pregnant mice. The early stage of gestation as GD 7 and 8 was found to be the most critical for the induction of various types of anomalies in the embryos. After treatment on these days, the highest percentages of gross and skeletal malformations were observed.
The types of malformations and their sites predominantly were the regions of head and face and were, in general, similar to those reported by earlier workers (36, 37, 38, 39).
It was evident that the specific action of OTA, during the early neurulation stage in embryonic development, was critical to induce anomalies, mostly in the craniofacial region.
The embryo defects observed in our experiments are in accord with the results obtained by Wei and Sulik in their magnificent work (14).
Wei and Sulik demonstrated, as after OTA treatment of pregnant mice at gestation day 7 - 8, the cell death, resulting from vital staining with Nile blue sulphate, is localized in selected cell population, and interestingly the area of major cell death was localized in the somatopleuric portion of the lateral plate mesoderm, that is the precursor of the body wall. As demonstrate by these authors staining was heavy in the frontonasal prominence, the region rostral to the developing eyes, when compared with staged untreated control. Some E16 embryos, furthermore, shown exencephaly while the craniofacial malformation, well evident at this stage, consist in a remarkable deformation or absence of nasal prominence derivative, frequently associated with midline cleft, and anterior neural tube closure defects, that proper derive from excessive cell death in the neuroepithelium and premigratory neural crest cells. The ocular structures frequently were reduced in size and closed positioned with hypotelorism or synoftalmia (14).
Being not clear the basis for the vulnerability of selected cell population to OTA, and the correlated teratogenic mechanism, we have presupposed the involvement of some Hox related gene that, if down or up expressed, can deregulate the correct gene cascade activation necessary for the differentiation program.
Dlx5 and Msx1 seem to play an important role in palatal formation and more in general in craniofacial differentiation, and for this motif we have studied the expression of these two genes in malformed embryos derived from mice treated with ochratoxin.
Also if Msx1 and Dlx5 act independently in the development of craniofacial skeleton (31) their expression in the developing head appears to be complementary. Msx proteins are mainly transcriptional repressors (40), while Dlx proteins are usually activators (41). For this reason, the possibility that the Msx1 protein may normally repress Dlx5 expression in the palate appeared likely. Since, the Dlx and Msx homeoproteins are known to form heterodimers in vitro and the interaction leads to abrogation of their DNA-binding and transcriptional activities (22), some of the phenotypes observed in Msx1 or in Dlx5 mutant animals could be due to altered activity of the cognate protein partner. On this basis the ochratoxin could interfere in the heterodimer formation.
Several mechanisms have been proposed for Dlx function. Dlx genes may instruct cell precursors of the palatal early in development (i.e. neural crest or arch ectomesenchyme, where they are expressed), or may control expression of secreted diffusable molecules, or the cleft is the consequence of a generalized deformation of the cranium. (31) .
Moreover a variety of molecules have been implicated in signalling during morphogenesis of facial primordia, including secreted molecules (Shh, Bmp, Wnt, Fgf) and transcription factors (Dlx, Otx, Msx, Gli and Tbx) (42, 43, 44) . Dlx5 could act directly as an inhibitor of bone morphogenetic protein expression as Bmp4, or could modulate Bmp function by regulating the expression of Bmp antagonists, such as Noggin, Chordin, or Follistatin. Bmp4 is expressed at sites of fusion between prominences of the head primordium, including the palate (45) .
In the mouse, palatal cleft is often associated with a down regulation of Bmp4 in the anterior palate. More precisely Bmp4 do not function alone but in concert with the others bone morphogenetic protein as Bmp2 and Bmp7 (46) .
As demonstrated by Acampora et al., disruption of Dlx genes causes palatal cleft (29, 30, 47), although disruption of Dlx5 leads to a less severe cleft, as compared to Msx1.
The widely described role of Dlx5 in the maxillofacial structures formation supports our results, in fact the widespread shutdown of Dlx5 gene in embryos, derived from pregnant mice treated with OTA, it seems closely related to the observed deformities.
These results support the hypothesis that Dlx5 gene is target for this toxin and the inhibition of its function, directly or indirectly, could be at origin of deformities caused by this mycotoxin. It remains to be demonstrated whether the OTA inhibits the transcription of Dlx5 binding to the promoter of this gene or acting on other genes, which in turn regulate the expression of Dlx5.
Our in situ hybridization experiments, concerning Msx1, did not reveal any change in the expression of this gene between control embryos and those derived from OTA treatment. This does not exclude the involvement of Msx1 gene in more earlier stage, even considering that at E16 stage, as well as E14 one, this gene is expressed at very low level. Moreover, since the proteins derived from Msx1 and Dlx5 form eterodimers, the reduced expression of Dlx5 could reduce the possibility of formation of these eterodimers, the molecules that are proper functionally active in regulating the development of maxillo-facial segment. Another hypothesis is that the phenotypic changes observed do not depend by Dlx5 expression inhibition, but by the resulting deregulation of the downstream genes, like those coding for bone morphogenetic proteins, specifically involved in the formation of the palate. A reduced expression of Dlx5 then could act indirectly by altering the expression of these effector proteins, that results in an alteration of this differentiation pattern.
Because of widespread contamination and increased incidence of ochratoxin in human food (48, 49) and the presence of OTA in human blood, cord blood samples from pregnant women, colostrum and milk, fetal exposure of OTA might pose a potential risk in pre-natal and post-natal life for the human infants (50, 51). On the basis of these considerations we think it is important to improve the studies on mycotoxins food contaminations and on the molecular mechanism involved in the determination of differentiation defects.
6. ACKNOWLEDGEMENTS
Margherita Napoletano and Davide Pennino have contributed equally to the work. The authors are very grateful to Mr. M. Cermola for excellent collaboration in microscopy work, to Mr. S. Baiano and Mr. F. Moscatiello for their skilful technical assistance. This study was financially supported in part by Institute of Genetics and Biophysics A. Buzzati Traverso C.N.R.
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Abbrevations: OTA: Ochratoxin A; Dlx: distal-les 5; Otx: orthodenticle ; Msx: muscle segment homeobox; Hox: homeotic gene; DIG: dioxigenin; Tbx: T- box gene; Bmp: bone morphogenetic protein; Shh: sonic hedgehog gene; Wnt: wongless gene; Fgf: Fibroblast Growth Factor.
Key words: Ochratoxin A; mycotoxin; mouse teratogenicity; craniofacial abnormalities; maxillary differentiation; Homeotic gene; Dlx5 gene; Msx1 gene
Send correspondence to: Francesco Morelli, Institute of Genetics and Biophysics A. Buzzati Traverso, Via P. Castellino, 111, 80131 Naples, Italy, Tel: 39081-6132252; Fax: 39081-6132627, E-mail:morelli@igb.cnr.it