Studio degli effetti neurotossici e genotossici dell’esposizione in vitro ed in vivo al Chlorpyrifos, un pesticida orgafosfato.

ABSTRACT Chlorpyrifos (CPF) is an organophosphate insecticide used primarily to control foliage and soilborn insect pests on a variety of food and feed crops. The main mechanism of CPF action is, likely the other organophoshates, the inhibition of acetylcholinesterase (AChE), which results in accumulation of acetylcholine and subsequent hyperactivity in the cholinergic system. However, multiple developmental studies on animal models have reported that chronic CPF exposure can alter brain development and neuronal morphogenesis even in the absence of significant AChE inhibition (Campbell et al., 1997; Das and Barone, 1999; Levin et al., 2002; Slotkin et al., 2006, 2007; Timofeeva et al., 2008a,b; Tussellino et al., 2016). In aquatic lower vertebrates, fish and amphibians, exposure to this compound has been correlated to teratogenic activity wich results in several anatomical alterations. In mammals, maternal exposure to CPF has been reported to induce dose-related abnormalities such as slower brain growth and cerebral cortex thinning. In mice, prenatal CPF exposure has been found to have the potential of affecting long-term brain cognitive function, but the mechanisms are not fully understood. On the other hand, several studies demonstrated that CPF can affect endocrine system, mainly thyroid and adrenal glands; moreover,it has been demonstrated that CPF has an inhibitory effect on the secretion of pituitary hormones such as luteinizing hormone (LH) and follicle stimulating hormone (FSH), and, in rat testis, can determine a decrease in testosterone biosynthesis. Based on these evidence, the aim of this PhD project is to gain a deeper understanding of the altered regulatory mechanisms triggered by environmental exposure to CPF. For this reason, we evaluated the effects of CPF on both aquatic and terrestrial model vertebrates, focusing, mainly, on 3 aspects: 1. Adverse effects during embryo development and neurogenesis in aquatic organisms: Xenopus laevis 2. Putative neurodegenerative alterations after long-lasting in utero and post-natally exposure: Mus musculus 3. Effects of CPF on mammalian sperm and fertility rate: Bos taurus Xenopus laevis In the first part of our study, we investigated the effects of exposure to different CPF concentrations (10, 15 and 20 mg/L) on Xenopus laevis embryos from stage 4/8 to stage 46. Some of the morphological changes we detected in CPF-exposed embryos included cranial neural crest cell (NCC)-derived structures. For this reason, we analyzed the expression of select genes involved in hindbrain patterning (egr2), cranial neural crest chondrogenesis, and craniofacial development (fgf8, bmp4, sox9, hoxa2 and hoxb2). We found that CPF exposure induced a reduction in transcription of all the genes involved in Cranial Neural Crest-dependent chondrogenesis, with largest reductions in fgf8 and sox9; whereas, in hindbrain, we did not find any alterations in egr2 expression. Thus, changes in the expression of fgf8, bmp4, and sox9, which are master regulators of several developmental pathways, result in teratogenic alterations of the facial skeleton. Interestingly, in birds and mammals (Le Douarin et al. 2007, the cranial NCC region forming the facial skeleton is also critical for the development of the anterior brain, and the interactions between these two regions during development require a correct balance of Fgf8 and of Bmp4 levels. The altered gene expression levels described in this work provide preliminary data that may help explain the correlation between OP and neurodevelopmental toxicity observed in mammals exposed to these molecules before birth (Chen et al., 2014). Mus musculus To study the effects of chronic CPF exposure in mammals we used F1 offspring (sacrificed at 3 and 8 months) of Mus musculus, exposed in utero and postnatally to 3 doses of CPF (0,1-1-10 mg/kg/d), commonly found on fruit and vegetables. The analysis was performed evaluating the alterations reported by 84 genes associated to Parkinson disease, using RT2 Profiler PCR Arrays. First of all, we evidenced a clear dose–response relationship in brains of 3 as well as 8 month mice exposed to the pesticide both for AChE inhibition as well as alterations of gene expression, although the panel of altered genes differed between the two aged groups. Overall, chlorpyrifos exposure produced a mixed pattern of up- and down-regulation, which is more evident in in the higher-CPF groups (10 mg/kg/d)). However, while there was still a dose response relationship, not apprenciable worsening was detectable with the increase of exposure time because brains of eldest mice showed a sort of general recovery of gene functionality with agenig. The total number of gene networks affected sharply reduced at 8 months and AchE values shift from highly inhibited, approximately 80% in 3 months, to 30% in the eldest mice. It was interesting to note that some of genes significantly and steadily down-regulated were directly related to Parkinson's onset, such as e Park 2, Sv2b, Gabbr2, Sept5, Atxn2. Interestingly, at 8 months we reported an up-regulation of the expression of Park7, HSPa4, Rgs4, and Chgb. Increased level of Park7 and HSPA4, considered neuroprotectors, together with the decrease of brain cholinesterase inhibition may, be explained as an adaptive response to the chronical stress induce by exposure to the pesticide and justify the recovery observed. Our evidence provide useful connections on the different scenarios determined by chronically environmental exposure to organophophate pesticides. Based on our experimental results, though animals do not show full blown syndrome, it is conceivable to hypothesize that their brain cells are more susceptible to develop specific neurological diseases. Bos Taurus Frozen semen samples were incubated with different concentrations of CPF (5,10,25,50μg/ml) for 2 h, and various spermatozoa functional parameters were assessed: motility, in vitro fertilization rates, DNA fragmentation and chromatin alterations, methylation pattern. Progressive forward motility was significantly (p 10 μg/ml). The vitro fertilization capabilities and the percentage of 8 cell embryos were significantly reduced at 50 μg/ml of chlorpyrifos when compared to control. Evaluation of the global sperm DNA methylation level, carried out at 10 and 25 μg/ml of CPF, showed no differences between the treated groups and the control. To confirm the results, we then selected 2 genes, Xist and GNAS, known to be imprinted in human and mice. In particular, we analyzed 10 CpG islands XIST promoter and 22 CpG islands for GNAS promoter. While, GNAS regolative region didn’t show any significative variations in methylation level, XIST promoter showed an increase in sperm DNA methylation after treatment (10ug/ml). The present study demonstrates that low doses of CPF impair sperm quality and can reduce male fertility potential. Moreover, our results showed that Chlorpyrifos exerted reproductive toxicology of sperm cells through various means, mainly altered sperm motiliy and reduction of fertilization rate. Conclusions On the basis of our experimental results, it is possible to assume that, subjects exposed daily to Chlorpyrifos, although not showing obvious signs of toxicity, may have higher sensitivity in developing pathologies, in particular neurological and reproductive disordes

Characterization of Two Transposable Elements and an Ultra-Conserved Element Isolated in the Genome of Zootoca vivipara (Squamata, Lacertidae)

: Transposable elements (TEs) constitute a considerable fraction of eukaryote genomes representing a major source of genetic variability. We describe two DNA sequences isolated in the lizard Zootoca vivipara, here named Zv516 and Zv817. Both sequences are single-copy nuclear sequences, including a truncation of two transposable elements (TEs), SINE Squam1 in Zv516 and a Tc1/Mariner-like DNA transposon in Zv817. FISH analyses with Zv516 showed the occurrence of interspersed signals of the SINE Squam1 sequence on all chromosomes of Z. vivipara and quantitative dot blot indicated that this TE is present with about 4700 copies in the Z. vivipara genome. FISH and dot blot with Zv817 did not produce clear hybridization signals. Bioinformatic analysis showed the presence of active SINE Squam 1 copies in the genome of different lacertids, in different mRNAs, and intronic and coding regions of various genes. The Tc1/Mariner-like DNA transposon occurs in all reptiles, excluding Sphenodon and Archosauria. Zv817 includes a trait of 284 bp, representing an amniote ultra-conserved element (UCE). Using amniote UCE homologous sequences from available whole genome sequences of major amniote taxonomic groups, we performed a phylogenetic analysis which retrieved Prototheria as the sister group of Metatheria and Eutheria. Within diapsids, Testudines are the sister group to Aves + Crocodylia (Archosauria), and Sphenodon is the sister group to Squamata. Furthermore, large trait regions flanking the UCE are conserved at family level

Cohesin mutations are synthetic lethal with stimulation of WNT signaling

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top 'hits' was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.Health Research Council of New Zealand (15/229) Julia A Horsfield Health Research Council of New Zealand (19/415) Ross D Hannan Julia A Horsfield Associazione Italiana per la Ricerca sul Cancro (IG23284) Antonio Musio The Maurice Wilkins centre for Molecular Biodiscovery (3705733) Jisha Antony Julia A Horsfiel

Temperature Incubation Influences Gonadal Gene Expression during Leopard Gecko Development

During development, sexual differentiation results in physiological, anatomical and metabolic differences that implicate not only the gonads but also other body structures. Sex in Leopard geckos is determined by egg incubation temperature. Based on the premise that the developmental decision of gender does not depend on a single gene, we performed an analysis on E. macularius to gain insights into the genes that may be involved in gonads' sexual differentiation during the thermosensitive period. All the genes were identified as differentially expressed at stage 30 during the labile phase of sex differentiation. In this way, the expression of genes known to be involved in gonadal sexual differentiation, such as WNT4, SOX9, DMRT1, Erα, Erβ, GnRH, P450 aromatase, PRLand PRL-R, was investigated. Other genes putatively involved in sex differentiation were sought by differential display. Our findings indicate that embryo exposure to a sex-determining temperature induces differential expression of several genes that are involved not only in gonadal differentiation, but also in several biological pathways (ALDOC, FREM1, BBIP1, CA5A, NADH5, L1 non-LTR retrotransposons, PKM). Our data perfectly fit within the new studies conducted in developmental biology, which indicate that in the developing embryo, in addition to gonadal differentiation, sex-specific tissue and metabolic polarization take place in all organisms

<i>Xenopus laevis</i> (Daudin, 1802) as a Model Organism for Bioscience: A Historic Review and Perspective

In vitro systems have been mainly promoted by authorities to sustain research by following the 3Rs principle, but continuously increasing amounts of evidence point out that in vivo experimentation is also of extreme relevance. Xenopus laevis, an anuran amphibian, is a significant model organism in the study of evolutionary developmental biology, toxicology, ethology, neurobiology, endocrinology, immunology and tumor biology; thanks to the recent development of genome editing, it has also acquired a relevant position in the field of genetics. For these reasons, X. laevis appears to be a powerful and alternative model to the zebrafish for environmental and biomedical studies. Its life cycle, as well as the possibility to obtain gametes from adults during the whole year and embryos by in vitro fertilization, allows experimental studies of several biological endpoints, such as gametogenesis, embryogenesis, larval growth, metamorphosis and, of course, the young and adult stages. Moreover, with respect to alternative invertebrate and even vertebrate animal models, the X. laevis genome displays a higher degree of similarity with that of mammals. Here, we have reviewed the main available literature on the use of X. laevis in the biosciences and, inspired by Feymann’s revised view, “Plenty of room for biology at the bottom”, suggest that X. laevis is a very useful model for all possible studies

Chlorpyrifos exposure affects fgf8, sox9, and bmp4 expression required for cranial neural crest morphogenesis and chondrogenesis in Xenopus laevis embryos

Chlorpyrifos (CPF) is an organophosphate insecticide used primarily to control foliage and soil-borne insect pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce dose-related abnormalities such as slower brain growth and cerebral cortex thinning. In lower vertebrates, for example, fish and amphibians, teratogenic activity of this compound is correlated with several anatomical alterations. Little is known about the effects of CPF on mRNA expression of genes involved in early development of the anatomical structures appearing abnormal in embryos. This study investigated the effects of exposure to different CPF concentrations (10, 15 and 20 mg/L) on Xenopus laevis embryos from stage 4/8 to stage 46. Some of the morphological changes we detected in CPF-exposed embryos included cranial neural crest cell (NCC)-derived structures. For this reason, we analyzed the expression of select genes involved in hindbrain patterning (egr2), cranial neural crest chondrogenesis, and craniofacial development (fgf8, bmp4, sox9, hoxa2 and hoxb2). We found that CPF exposure induced a reduction in transcription of all the genes involved in NCC-dependent chondrogenesis, with largest reductions in fgf8 and sox9; whereas, in hindbrain, we did not find any alterations in egr2 expression. Changes in the expression of fgf8, bmp4, and sox9, which are master regulators of several developmental pathways, have important implications. If these changes are confirmed to belong to a general pattern of alterations in vertebrates prenatally exposed to OP, they might be useful to assess damage during vertebrate embryo development. Environ. Mol. Mutagen., 2016. © 2016 Wiley Periodicals, Inc

Short interspersed DNA elements and miRNAs: a novel hidden gene regulation layer in zebrafish?

In this study, we investigated by in silico analysis the possible correlation between microRNAs (miRNAs) and Anamnia V-SINEs (a superfamily of short interspersed nuclear elements), which belong to those retroposon families that have been preserved in vertebrate genomes for millions of years and are actively transcribed because they are embedded in the 3' untranslated region (UTR) of several genes. We report the results of the analysis of the genomic distribution of these mobile elements in zebrafish (Danio rerio) and discuss their involvement in generating miRNA gene loci. The computational study showed that the genes predicted to bear V-SINEs can be targeted by miRNAs with a very high hybridization E-value. Gene ontology analysis indicates that these genes are mainly involved in metabolic, membrane, and cytoplasmic signaling pathways. Nearly all the miRNAs that were predicted to target the V-SINEs of these genes, i.e., miR-338, miR-9, miR-181, miR-724, miR-735, and miR-204, have been validated in similar regulatory roles in mammals. The large number of genes bearing a V-SINE involved in metabolic and cellular processes suggests that V-SINEs may play a role in modulating cell responses to different stimuli and in preserving the metabolic balance during cell proliferation and differentiation. Although they need experimental validation, these preliminary results suggest that in the genome of D. rerio, as in other TE families in vertebrates, the preservation of V-SINE retroposons may also have been favored by their putative role in gene network modulation

Chlorpyrifos effects on Xenopus laevis embryo development

Disorders of neurobehavioral development have been dramatically increased worldwide during the past decades and recent data highlighted that more than 10-15% of new-borns are affected by them with a prevalence rate of autism spectrum disorder and other cognitive impairments. An increased risk of attention-deficit hyperactivity disorders have been linked to prenatal exposure to Organophosphates pesticides (OPs), which are widely used on food crops grown in the EU. While they have been banned from indoor use, they are still the most prevalent pesticides in the EU, with Chlorpyrifos (CP) being the most commonly applied. Cohort studies provided new evidence that prenatal exposure to OPs can cause developmental neurotoxicity. In fact, younger individuals may be more susceptible than adults due to biological factors and exposure settings. In particular, maternal exposure to CP was found to be dose-related to slower brain growth and associated to several structural abnormalities such as thinning of cerebral cortex. It has been demonstrated that sub-lethal doses of neurotoxic pesticides affect behaviour by inhibiting acetylcholinesterase (AChE). While the cognitive short-term effects may be directly attributed to this inhibition, the mechanisms that underlie OP's long-term cognitive effects remain controversial and poorly understood. To elucidate through what mechanism CP induces alterations in neurogenesis, we used Xenopus laevis, an aquatic organism whose development occurs outside the mother’s body and that allowing monitoring anlagen morphogenesis in detail. We investigated CP effects on embryonic survival, histological alterations and the occurance of gross anatomical abnormalities. Briefly, Xenopus embryos, (stage 4/8) were bred in presence of a wide range of CP concentrations (0.01 mg/L- 25 mg/L). Preliminary results seem to indicate that CP can induce alterations possibly correlated to incorrect migration of neural crest cells. We also evaluated the expression profiles of several genes involved in neural development such as rax1, pax6 and fgf8

Brain Gene Expression is Influenced by Incubation Temperature During Leopard Gecko (Eublepharis macularius) Development

Sexual differentiation (SD) during development results in anatomical, metabolic, and physiological differences that involve not only the gonads, but also a variety of other biological structures, such as the brain, determining differences in morphology, behavior, and response in the breeding season. In many reptiles, whose sex is determined by egg incubation temperature, such as the leopard gecko, Eublepharis macularius, embryos incubated at different temperatures clearly differ in the volume of brain nuclei that modulate behavior. Based on the premise that "the developmental decision of gender does not flow through a single gene", we performed an analysis on E. macularius using three approaches to gain insights into the genes that may be involved in brain SD during the thermosensitive period. Using quantitative RT-PCR, we studied the expression of genes known to be involved in gonadal SD such as WNT4, SOX9, DMRT1, Erα, Erβ, GnRH, P450 aromatase, PRL, and PRL-R. Then, further genes putatively involved in sex dimorphic brain differentiation were sought by differential display (DDRT-PCR) and PCR array. Our findings indicate that embryo exposure to different sex determining temperatures induces differential expression of several genes that are involved not only in gonadal differentiation (PRL-R, Wnt4, Erα, Erβ, p450 aromatase, and DMRT1), but also in neural differentiation (TN-R, Adora2A, and ASCL1) and metabolic pathways (GP1, RPS15, and NADH12). These data suggest that the brains of SDT reptiles might be dimorphic at birth, thus behavioral experiences in postnatal development would act on a structure already committed to male or female

Specific Effects of Chronic Dietary Exposure to Chlorpyrifos on Brain Gene Expression— A Mouse Study

Chlorpyrifos (CPF) is an organophosphate insecticide used to control pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce cerebral cortex thinning, alteration of long-term brain cognitive function, and Parkinson-like symptoms, but the mechanisms of these processes are not fully understood. In this study, we aimed to gain a deeper understanding of the alterations induced in the brains of mice chronically exposed to CPF by dietary intake. For our purpose, we analysed F1 offspring (sacrificed at 3 and 8 months) of Mus musculus, treated in utero and postnatally with 3 different doses of CPF (0.1-1-10 mg/kg/day). Using RT2 Profiler PCR Arrays, we evaluated the alterations in the expression of 84 genes associated with neurodegenerative diseases. In the brains of exposed mice, we evidenced a clear dose–response relationship for AChE inhibition and alterations of gene expression. Some of the genes that were steadily down-regulated, such as Pink1, Park 2, Sv2b, Gabbr2, Sept5 and Atxn2, were directly related to Parkinson’s onset. Our experimental results shed light on the possibility that long-term CPF exposure may exert membrane signalling alterations which make brain cells more susceptible to develop neurodegenerative diseases