The heavy metal cadmium is a widespread toxic pollutant, released into the environment mainly by anthropogenic activities. Human exposure can occur through different sources: occupationally or environmentally, with its uptake through inhalation of polluted air, cigarette smoking or ingestion of contaminated food and water. It mainly enters the human body through the respiratory and the gastrointestinal tract and it accumulates in liver and kidneys. Brain is also a target of cadmium toxicity, since this toxicant may enter the central nervous system by increasing blood brain barrier permeability or through the olfactory nerves. In fact, cadmium exposure has been related to impaired functions of the nervous system and to neurodegenerative diseases, like amyotrophic lateral sclerosis (ALS). ALS is a fatal motor neuron pathology with the 90-95% of ALS cases being sporadic (sALS), while the remaining 5-10% of familial onset (fALS); among fALS, the 15-20% is attributed to mutations in superoxide dismutase 1 (SOD1). SOD1 is an antioxidant protein responsible for superoxide anions disruption and it is a homodimeric metalloenzyme of 32 kDa mainly located in the cytoplasm, with each monomer binding one catalytic copper ion and one structural zinc ion within a disulfide bonded conformer. Since oxidative stress is one of the major mechanisms of cadmium induced toxicity and an alteration of oxidative homeostasis, through depletion of antioxidant defences, is responsible for a plethora of adverse outcoming mainly leading to cell death; we focused on cadmium effect (1) on the energetic metabolism in human neuroblastoma SH-SY5Y cell line, (2) on the oxidative defences responses in differentiated human LUHMES neural cell line and (3) on the function of human SOD1 in a three models approach (recombinant protein in E. coli, in SH-SY5Y cell line and in the nematode Caenorhabditis elegans). The evaluation of energetic metabolism of SH-SY5Y neural cells treated with sub-lethal CdCl2 doses for 24 hours, showed an increase in glycolysis compared to control. This shift to anaerobic metabolism has been confirmed by both glycolytic parameters and greater ATP production from glycolysis than oxidative phosphorylation, index of less mitochondrial functionality in cadmium treated cells. Regarding the fuel oxidation cadmium caused an increase in glutamine dependency and a specular reduction in the fatty acids one, without altering the glucose dependency. Moreover, we observed an increase in total GSH, in the GSSG/GSH ratio and in lipid peroxidation, all index of an altered oxidative homeostasis better investigated in LUHMES cells. In this model a 24h cadmium administration enhanced the total GSH content at the lower doses, at which also activates Nrf2 through a better protein stabilization via p21 and P-Akt. The metal adverse effects on cell viability can be rescued by GSH addition and by cadmium treatment in astrocytes- or microglia-conditioned medium. In the latter cases the total GSH level remains comparable to untreated cells even at higher CdCl2 concentrations. Finally, SOD1 catalytical activity has been investigated in the presence of cadmium. The first evaluation of this metal combined with fixed copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21, showed a dose-dependent reduction in SOD1 activity only when copper is added to cellular medium, while the expression remains always constant. Similar results were obtained in SH-SY5Y cell line, in which SOD1 enzymatic activity decreased in a dose- and time-dependent way after cadmium treatment for 24 and 48 hours, without altering its expression; as well as in the Caenorhabditis elegans model, where a 16 hours cadmium treatment caused a 25% reduction only in SOD1 activity. In conclusion, cadmium caused a shift to anaerobiosis, a Nrf2 activation, with increased GSH production, and a reduction in SOD1 activity.

Il cadmio, elemento chimico ampiamente usato in ambito industriale, è considerato un contaminante ambientale con effetti tossici sugli organismi viventi. Il suo ingresso nel corpo umano può avvenire per inalazione o ingestione di cibi ed acqua contaminati, fumo di sigaretta o impiego professionale, con tratto respiratorio e gastrointestinale principalmente coinvolti nel suo assorbimento cellulare. Anche il cervello è un bersaglio della tossicità del cadmio, che può entrare nel sistema nervoso centrale tramite una maggiore permeabilità della barriera ematoencefalica o attraverso i nervi olfattivi. Infatti, l'esposizione al cadmio è stata correlata sia ad alterazioni funzionali del sistema nervoso sia a malattie neurodegenerative, come la sclerosi laterale amiotrofica (SLA). Il 90-95% dei casi di SLA sono sporadici (sALS), mentre il restante 5-10% ha origine familiare (fALS), di cui il 15-20% è attribuito a mutazioni nel gene dell’enzima antiossidante superossido dismutasi 1 (SOD1). SOD1 è un omodimero di 32 kDa, in cui ciascun monomero presenta un ponte disulfuro e due ioni metallici, il rame con ruolo catalitico e lo zinco con funzione strutturale. Poiché uno dei principali meccanismi con cui il cadmio esercita la propria tossicità è lo stress ossidativo, responsabile di un insieme di eventi avversi che culminano nella morte cellulare, scopo di questa tesi è lo studio dell'effetto neurotossico del cadmio sul metabolismo energetico nella linea cellulare umana SH-SY5Y, sulle difese antiossidanti in cellule LUHMES differenziate e sulla funzione di SOD1 in tre modelli sperimentali (proteina ricombinante in E. coli, linea cellulare SH-SY5Y e nematode Caenorhabditis elegans). La valutazione del metabolismo energetico in cellule SH-SY5Y trattate per 24 ore con dosi sub-letali di CdCl2 ha evidenziato il passaggio ad un metabolismo anaerobico; infatti cellule trattate mostrano un aumento della glicolisi, una maggiore produzione di ATP per via glicolitica e una ridotta funzionalità mitocondriale rispetto al controllo. L’apporto bioenergetico in presenza di cadmio non altera la dipendenza da glucosio, ma aumenta quella da glutammina riducendo l’apporto derivato dagli acidi grassi. Inoltre, si osserva un aumento del GSH totale, del rapporto GSSG/GSH e della perossidazione lipidica, tutti indici di un'alterata omeostasi ossidativa. Quest’ultima è stata investigata in cellule LUHMES differenziate, in cui 24 ore di esposizione al cadmio hanno determinato, alle dosi più basse, un aumento del livello di GSH totale e un’attivazione di Nrf2 mediata da p21 e P-Akt. Gli effetti negativi del cadmio sulla vitalità cellulare possono essere annullati dall'aggiunta di GSH e dal trattamento in conditioned medium (CM) ottenuto da astrociti o microglia. Nelle LUHMES trattate in CM il livello totale di GSH rimane paragonabile a quello delle cellule non trattate anche alle concentrazioni più elevate di CdCl2. Infine, l’effetto del cadmio, combinato a dosi fisse di rame e/o zinco, sull'attività catalitica della proteina ricombinante GST-SOD1, espressa in E. coli BL21, ha mostrato una riduzione dose-dipendente dell'attività di SOD1 solo in presenza di rame, mentre il livello di espressione proteica rimane sempre costante. Risultati analoghi sono stati ottenuti nella linea cellulare SH-SY5Y, in cui l'attività enzimatica di SOD1 è diminuita in modo sia dose che tempo-dipendente dopo il trattamento con cadmio per 24 e 48 ore, così come nel nematode C. elegans, in cui si osserva una riduzione del 25% nell’attività di SOD1 dopo 16 ore di trattamento con cadmio. In entrambi i casi il livello di espressione proteica dell’enzima rimane invariato. In conclusione, il cadmio ha determinato il passaggio ad un metabolismo più anaerobico, l'attivazione di Nrf2, con conseguente aumento nella produzione di GSH e una riduzione dell'attività di SOD1.

(2021). The cadmium altered oxidative homeostasis leads to energetic metabolism rearrangement, Nrf2 activation with increased GSH production and reduced SOD1 activity in neural cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2021).

The cadmium altered oxidative homeostasis leads to energetic metabolism rearrangement, Nrf2 activation with increased GSH production and reduced SOD1 activity in neural cells

BOVIO, FEDERICA
2021

Abstract

The heavy metal cadmium is a widespread toxic pollutant, released into the environment mainly by anthropogenic activities. Human exposure can occur through different sources: occupationally or environmentally, with its uptake through inhalation of polluted air, cigarette smoking or ingestion of contaminated food and water. It mainly enters the human body through the respiratory and the gastrointestinal tract and it accumulates in liver and kidneys. Brain is also a target of cadmium toxicity, since this toxicant may enter the central nervous system by increasing blood brain barrier permeability or through the olfactory nerves. In fact, cadmium exposure has been related to impaired functions of the nervous system and to neurodegenerative diseases, like amyotrophic lateral sclerosis (ALS). ALS is a fatal motor neuron pathology with the 90-95% of ALS cases being sporadic (sALS), while the remaining 5-10% of familial onset (fALS); among fALS, the 15-20% is attributed to mutations in superoxide dismutase 1 (SOD1). SOD1 is an antioxidant protein responsible for superoxide anions disruption and it is a homodimeric metalloenzyme of 32 kDa mainly located in the cytoplasm, with each monomer binding one catalytic copper ion and one structural zinc ion within a disulfide bonded conformer. Since oxidative stress is one of the major mechanisms of cadmium induced toxicity and an alteration of oxidative homeostasis, through depletion of antioxidant defences, is responsible for a plethora of adverse outcoming mainly leading to cell death; we focused on cadmium effect (1) on the energetic metabolism in human neuroblastoma SH-SY5Y cell line, (2) on the oxidative defences responses in differentiated human LUHMES neural cell line and (3) on the function of human SOD1 in a three models approach (recombinant protein in E. coli, in SH-SY5Y cell line and in the nematode Caenorhabditis elegans). The evaluation of energetic metabolism of SH-SY5Y neural cells treated with sub-lethal CdCl2 doses for 24 hours, showed an increase in glycolysis compared to control. This shift to anaerobic metabolism has been confirmed by both glycolytic parameters and greater ATP production from glycolysis than oxidative phosphorylation, index of less mitochondrial functionality in cadmium treated cells. Regarding the fuel oxidation cadmium caused an increase in glutamine dependency and a specular reduction in the fatty acids one, without altering the glucose dependency. Moreover, we observed an increase in total GSH, in the GSSG/GSH ratio and in lipid peroxidation, all index of an altered oxidative homeostasis better investigated in LUHMES cells. In this model a 24h cadmium administration enhanced the total GSH content at the lower doses, at which also activates Nrf2 through a better protein stabilization via p21 and P-Akt. The metal adverse effects on cell viability can be rescued by GSH addition and by cadmium treatment in astrocytes- or microglia-conditioned medium. In the latter cases the total GSH level remains comparable to untreated cells even at higher CdCl2 concentrations. Finally, SOD1 catalytical activity has been investigated in the presence of cadmium. The first evaluation of this metal combined with fixed copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21, showed a dose-dependent reduction in SOD1 activity only when copper is added to cellular medium, while the expression remains always constant. Similar results were obtained in SH-SY5Y cell line, in which SOD1 enzymatic activity decreased in a dose- and time-dependent way after cadmium treatment for 24 and 48 hours, without altering its expression; as well as in the Caenorhabditis elegans model, where a 16 hours cadmium treatment caused a 25% reduction only in SOD1 activity. In conclusion, cadmium caused a shift to anaerobiosis, a Nrf2 activation, with increased GSH production, and a reduction in SOD1 activity.
FUSI, PAOLA ALESSANDRA
LOTTI, MARINA
cadmio; cellule neuronali; SOD1; neurotossicità; C. elegans
cadmium; neural cells; SOD1; neurotoxicity; C. elegans
BIO/10 - BIOCHIMICA
English
30-mar-2021
TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi)
33
2019/2020
open
(2021). The cadmium altered oxidative homeostasis leads to energetic metabolism rearrangement, Nrf2 activation with increased GSH production and reduced SOD1 activity in neural cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2021).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/309982
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