Pallotta, Maria Michela (2017) Studio degli effetti neurotossici e genotossici dell’esposizione in vitro ed in vivo al Chlorpyrifos, un pesticida orgafosfato. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: Italiano
Title: Studio degli effetti neurotossici e genotossici dell’esposizione in vitro ed in vivo al Chlorpyrifos, un pesticida orgafosfato.
Pallotta, Maria
Date: 8 April 2017
Number of Pages: 126
Institution: Università degli Studi di Napoli Federico II
Department: Biologia
Dottorato: Biologia
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
Carotenuto, RosaUNSPECIFIED
Date: 8 April 2017
Number of Pages: 126
Keywords: Effetti del Chlorpyrifos
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/06 - Anatomia comparata e citologia
Date Deposited: 20 Apr 2017 11:38
Last Modified: 16 Mar 2018 13:06
DOI: 10.6093/UNINA/FEDOA/11641

Collection description

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 < 0.05) reduced in spermatozoa exposed to the 10, 25,50 μg/ml of CPF while DNA fragmentation and putative chromatin deconstruction appeared to increase significantly (p < 0.05) only at CPF higher concentrations (>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.


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