The aim of my thesis was to evaluate the impact of per-and polyfluoroalkyl substances (PFAS) exposure on the skeletal development both in healthy condition as well as in presence of the bone disease osteogenesis imperfecta (OI) using zebrafish models. OI is a heterogeneous group of skeletal disorders characterized by low bone mass and reduced bone strength, resulting in skeletal fragility and easy susceptibility to fractures, bone deformity and growth deficiency. The majority of OI cases have autosomal dominant inheritance and are caused by mutations in either COL1A1 or COL1A2 genes, coding for α1 or α2 chains of type I collagen. These mutations cause structural or quantitative defects in type I collagen and cause a skeletal phenotype ranging from mild to lethal. Per-and polyfluoroalkyl substances (PFAS) are a group of over 4700 fluorinated synthetic compounds widely used in commercial and industrial products due to their crucial features, like high stability to chemical and thermal degradation. PFAS are extremely permanent in the environment and tend to bioaccumulate and biomagnificate in the organism, causing a wide range of negative health effects, representing a serious concern for both wildlife and human health. Thus, to evaluate the impact of heptadecafluorooctanesulfonic acid tetraethylammonium salt (PFOS) and perfluorooctanoic acid (PFOA) on fragile skeletal condition, the dominant OI zebrafish Chihuahua (Chi/+) was used. Zebrafish (Danio rerio) shares the same bone cell types and ossification processes with mammals, which make it a valid model for studying skeletal diseases. WT;Tg(OlSp7:nlsGFP)zf132 and Chi/+;Tg(OlSp7:nlsGFP)zf132 larvae characterized by GFP expression under the Sp7 promoter allowed to follow osteoblasts (OBs) in vivo. Alizarin red staining allowed to detect mineral deposition. Interestingly, PFOS exposure significantly reduced Sp7GFP+ delimited operculum area in both WT and Chi/+ compared to untreated larvae. Also, a reduced mineral deposition was detected in WT exposed to PFOS compared to untreated WT, whereas no changes were evident in Chi/+ group. Real time qPCR revealed an increase in the expression of the anti-apoptotic gene bcl-2 in both WT and Chi/+ grown in presence of PFOS, suggesting an attempt to rescue a possible apoptotic stimulation due to the exposure. In mutant fish the already reduced expression of the early marker for pre-osteoblasts runx2a got slightly worse after PFOS exposure, thus confirming the negative impact of pollutant on bone development. A strong effect on bone formation was also demonstrated by PFOA exposure. Indeed, a significant reduction in Sp7GFP+ OBs delimited area was detected in Chi/+ treated compared to untreated control larvae. Also, a reduction in the mineralized area was observed in both WT and Chi/+ after PFOA administration. In conclusion, our data suggested that PFOS and PFOA exposure impair operculum bone area deposited by OBs and/or mineral deposition in a molecule-specific manner. The gene expression analysis also indicated an impact on cell activity induced by PFOS exposure in both genotypes, thus confirming the negative effect of pollutants in both physiological and pathological conditions.
L’obiettivo della mia tesi è stato quello di valutare l’impatto che l’esposizione a sostanze per- e polifluoroalchiliche (PFAS) ha sul corretto sviluppo osseo sia in condizioni fisiologiche che patologiche. In particolare, mi sono focalizzata sullo studio dell’effetto dei PFAS in un modello zebrafish di osteogenesi imperfetta a trasmissione dominante, chiamato Chihuahua. L’osteogenesi imperfetta è una patologia scheletrica caratterizzata da una ridotta massa e resistenza ossea che provoca fragilità, deformità ossea e difetti della crescita. La maggior parte dei casi di OI sono causati da mutazioni a trasmissione dominante nei geni COL1A1 o COL1A2, che codificano rispettivamente per la catena α1 e α2 del collagene di tipo I. Queste mutazioni causano difetti strutturali e difetti quantitativi nel collagene di tipo I portando a un fenotipo scheletrico la cui gravità varia da lieve a letale. Le sostanze per- e polifluoroalchiliche (PFAS) rappresentano più di 4700 composti sintetici largamente utilizzati nella produzione industriale e in prodotti commerciali a seguito di alcune caratteristiche di fondamentale importanza, come l’alta resistenza alla degradazione chimica e termica. I PFAS permangono a lungo nell’ambiente e tendono ad accumularsi nell’organismo, causando un’ampia gamma di effetti negativi sulla salute dell’uomo. Zebrafish (Danio rerio) ha il 70% dei geni omologhi a quelli umani, inoltre è caratterizzato dalle stesse cellule ossee dei mammiferi e dagli stessi processi di ossificazione. Per questi motivi è un valido modello per lo studio di patologie ossee. Durante il mio tirocinio di tesi sono state utilizzate larve WT;Tg(OlSp7:nlsGFP)zf132 e Chi/+;Tg(OlSp7:nlsGFP)zf132, che esprimono la GFP sotto il promotore di osterix (sp7), marcatore specifico degli osteoblasti immaturi. Grazie all’uso del transgenico e grazie alla colorazione con rosso di alizarina, specifica per il minerale, è stato possibile analizzare l’area dell’opercolo delimitata dagli osteoblasti immaturi sp7GFP+) e contemporaneamente valutare la qualità di minerale depositato. L’esposizione al PFOS ha ridotto in maniera significativa l’area dell’opercolo delimitata dagli osteoblasti sp7GFP+ sia nei WT che nei Chi/+ rispetto alle larve di controllo non trattate. È stata osservata anche una ridotta deposizione di minerale nelle larve WT esposte al PFOS rispetto ai controlli. La Real time qPCR ha evidenziato un aumento di espressione del gene anti-apoptotico bcl-2 sia nei WT che nei Chi/+ esposti al PFOS, nel probabile tentativo di contrastare l’attivazione dell’apoptosi in seguito all’esposizione all’inquinante. Nei Chi/+ l’espressione di runx2a, marker precoce dei pre-osteoblasti, è inferiore rispetto ai WT e si riduce ulteriormente dopo l’esposizione al PFOS, anche se non si raggiunge la significatività, confermando l’effetto negativo che questa molecola ha sullo sviluppo osseo. Anche l’esposizione al PFOA ha causato un forte impatto sullo sviluppo osseo, in quanto ha portato ad una riduzione significativa dell’area dell’opercolo delimitata degli osteoblasti sp7GFP+ nei Chi/+ esposti rispetto ai Chi/+ controllo. Inoltre, una ridotta mineralizzazione ossea è stata evidenziata sia nei WT che nei Chi/+ dopo esposizione a PFOA. In conclusione, i nostri dati suggeriscono che l’esposizione ai PFAS provochi una compromissione della formazione ossea a livello dell’opercolo del processo di mineralizzazione sia in condizioni patologiche che fisiologiche. I livelli di espressione di geni legati all’apoptosi e al differenziamento degli osteoblasti confermano l’effetto negativo di questi composti sia nelle larve WT che in quelle mutate.
Le sostanze perfluoroalchiliche impattano negativamente sullo sviluppo scheletrico in un modello zebrafish di osteogenesi imperfetta
RIVA, ELEONORA
2022/2023
Abstract
The aim of my thesis was to evaluate the impact of per-and polyfluoroalkyl substances (PFAS) exposure on the skeletal development both in healthy condition as well as in presence of the bone disease osteogenesis imperfecta (OI) using zebrafish models. OI is a heterogeneous group of skeletal disorders characterized by low bone mass and reduced bone strength, resulting in skeletal fragility and easy susceptibility to fractures, bone deformity and growth deficiency. The majority of OI cases have autosomal dominant inheritance and are caused by mutations in either COL1A1 or COL1A2 genes, coding for α1 or α2 chains of type I collagen. These mutations cause structural or quantitative defects in type I collagen and cause a skeletal phenotype ranging from mild to lethal. Per-and polyfluoroalkyl substances (PFAS) are a group of over 4700 fluorinated synthetic compounds widely used in commercial and industrial products due to their crucial features, like high stability to chemical and thermal degradation. PFAS are extremely permanent in the environment and tend to bioaccumulate and biomagnificate in the organism, causing a wide range of negative health effects, representing a serious concern for both wildlife and human health. Thus, to evaluate the impact of heptadecafluorooctanesulfonic acid tetraethylammonium salt (PFOS) and perfluorooctanoic acid (PFOA) on fragile skeletal condition, the dominant OI zebrafish Chihuahua (Chi/+) was used. Zebrafish (Danio rerio) shares the same bone cell types and ossification processes with mammals, which make it a valid model for studying skeletal diseases. WT;Tg(OlSp7:nlsGFP)zf132 and Chi/+;Tg(OlSp7:nlsGFP)zf132 larvae characterized by GFP expression under the Sp7 promoter allowed to follow osteoblasts (OBs) in vivo. Alizarin red staining allowed to detect mineral deposition. Interestingly, PFOS exposure significantly reduced Sp7GFP+ delimited operculum area in both WT and Chi/+ compared to untreated larvae. Also, a reduced mineral deposition was detected in WT exposed to PFOS compared to untreated WT, whereas no changes were evident in Chi/+ group. Real time qPCR revealed an increase in the expression of the anti-apoptotic gene bcl-2 in both WT and Chi/+ grown in presence of PFOS, suggesting an attempt to rescue a possible apoptotic stimulation due to the exposure. In mutant fish the already reduced expression of the early marker for pre-osteoblasts runx2a got slightly worse after PFOS exposure, thus confirming the negative impact of pollutant on bone development. A strong effect on bone formation was also demonstrated by PFOA exposure. Indeed, a significant reduction in Sp7GFP+ OBs delimited area was detected in Chi/+ treated compared to untreated control larvae. Also, a reduction in the mineralized area was observed in both WT and Chi/+ after PFOA administration. In conclusion, our data suggested that PFOS and PFOA exposure impair operculum bone area deposited by OBs and/or mineral deposition in a molecule-specific manner. The gene expression analysis also indicated an impact on cell activity induced by PFOS exposure in both genotypes, thus confirming the negative effect of pollutants in both physiological and pathological conditions.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/16914