The Cytochrome P450 isozymes involved in xenobiotic metabolism are ubiquitous enzymes, predominantly expressed in the liver in comparison to the extrahepatic tissues. In the brain, CYP expression is approximately 0.5-2% of that in liver microsomes, and most of the isoforms appears to be very low for playing a role in overall total body clearance. Brain basal expression and up-regulation can however significantly affect local disposition of xenobiotic or endogenous compounds. Several reports, in fact, indicate that environmental toxins may play a role in the pathogenesis of neurodegenerative disorders by directly damaging neurons or by CYPs-mediated their bioactivation into toxic compounds. Among the different isoforms, CYP2D6 is particularly involved in the metabolism of exogenous drugs, neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, which selectively target nigrostriatal dopaminergic pathways), as well as endogenous compounds, including dopamine. Moreover, CYP2D6 extended polymorphism is significantly associated with an increased PD risk, owing to a lower capability in the metabolism of neurotoxic compounds such as pesticides. On the contrary, other studies did not support the association between PD and the poor CYP2D6-dependent metabolism, thus suggesting that PD is most likely the result of interactions between multiple genetic and environmental factors. The aim of the present study was to increase the knowledge on the role of neuronal CYP-dependent oxidative metabolism and to clarify whether it might affect xenobiotic-promoted neurodegeneration by using an in vitro model based on human neuroblastoma-derived SH-SY5Y cells. The first step was to promote the differentiation of neuroblastoma cells into mature human dopaminergic neurons phenotype. Two different protocols based on retinoic acid and phorbol ester or retinoic acid and brain derived neurotrophic factor were set up. The switch into dopaminergic phenotype was assessed by studying the cell morphology and the expression of the most important proteins recognized as neuronal biomarkers. Results showed that after differentiation, SH-SY5Y cells demonstrated extensive and elongated neuritic projections, a significant increased content of NeurineN, Synaptophysin, and β-tubulin III, as well as dopamine transporter. Two well know inducers such as β-naphtoflavone (βNF) and ethanol (EtOH) were then used for promoting CYPs induction in both undifferentiated (UD) and differentiated SH-SY5Y cells. qRT-PCR analysis showed that both compounds significantly increased the mRNA expression of CYP2E1 and CYP2D6 isoforms in all SH-SY5Y cells population, while 1A1 and 3A4 were substantially unaffected. 7 To explore the role of CYP in the protection toward neurotoxicity, SH-SY5Y cells possessing increased CYPs content were treated with 1-methyl-4-phenylpyridinium (MPP+), the MAO-B-dependent toxic metabolite of MPTP, or rotenone, two neurotoxins widely used to reproduce PD models in vivo and in vitro. The treatment with EtOH and βNF protected UD cells against MPP+ toxicity, whereas only βNF partially reverted the cytotoxic insult promoted by Rotenone. Moreover, the differentiated SH-SY5Y cells resulted to be less sensitive to the cytotoxic effects caused by the neurotoxins and consequently less responsive to the protection promoted by the CYPs induction. Furthermore, both βNF and EtOH treatments partially reverted the loss in mitochondria membrane potential, complex I impairment, as well as the increased ROS formation when UD were exposed to MPP+ or rotenone. Taken together, these results support the possible role of CYP isoforms in the neuroprotection against xenobiotic insult, especially as far as concerned the metabolic activity of CYP2D6 and CYP2E1. However, it would be interesting to investigate the induction dynamics of the CYPs here analysed and to understand the pathways triggered by βNF and EtOH leading to CYP induction. In conclusion, the impact that these isoforms have in the metabolism of other drugs, together with the present results, bring new insights on the role of brain CYPs in the effects of drugs and can drive future therapeutic approaches for ameliorating the therapy of neurological diseases.

Chiaino, E. (2022). CYP450 expression and regulation in SH-SY5Y cells. Possible role in neurotoxin-mediated injury.

CYP450 expression and regulation in SH-SY5Y cells. Possible role in neurotoxin-mediated injury

Chiaino, Elda
2022

Abstract

The Cytochrome P450 isozymes involved in xenobiotic metabolism are ubiquitous enzymes, predominantly expressed in the liver in comparison to the extrahepatic tissues. In the brain, CYP expression is approximately 0.5-2% of that in liver microsomes, and most of the isoforms appears to be very low for playing a role in overall total body clearance. Brain basal expression and up-regulation can however significantly affect local disposition of xenobiotic or endogenous compounds. Several reports, in fact, indicate that environmental toxins may play a role in the pathogenesis of neurodegenerative disorders by directly damaging neurons or by CYPs-mediated their bioactivation into toxic compounds. Among the different isoforms, CYP2D6 is particularly involved in the metabolism of exogenous drugs, neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, which selectively target nigrostriatal dopaminergic pathways), as well as endogenous compounds, including dopamine. Moreover, CYP2D6 extended polymorphism is significantly associated with an increased PD risk, owing to a lower capability in the metabolism of neurotoxic compounds such as pesticides. On the contrary, other studies did not support the association between PD and the poor CYP2D6-dependent metabolism, thus suggesting that PD is most likely the result of interactions between multiple genetic and environmental factors. The aim of the present study was to increase the knowledge on the role of neuronal CYP-dependent oxidative metabolism and to clarify whether it might affect xenobiotic-promoted neurodegeneration by using an in vitro model based on human neuroblastoma-derived SH-SY5Y cells. The first step was to promote the differentiation of neuroblastoma cells into mature human dopaminergic neurons phenotype. Two different protocols based on retinoic acid and phorbol ester or retinoic acid and brain derived neurotrophic factor were set up. The switch into dopaminergic phenotype was assessed by studying the cell morphology and the expression of the most important proteins recognized as neuronal biomarkers. Results showed that after differentiation, SH-SY5Y cells demonstrated extensive and elongated neuritic projections, a significant increased content of NeurineN, Synaptophysin, and β-tubulin III, as well as dopamine transporter. Two well know inducers such as β-naphtoflavone (βNF) and ethanol (EtOH) were then used for promoting CYPs induction in both undifferentiated (UD) and differentiated SH-SY5Y cells. qRT-PCR analysis showed that both compounds significantly increased the mRNA expression of CYP2E1 and CYP2D6 isoforms in all SH-SY5Y cells population, while 1A1 and 3A4 were substantially unaffected. 7 To explore the role of CYP in the protection toward neurotoxicity, SH-SY5Y cells possessing increased CYPs content were treated with 1-methyl-4-phenylpyridinium (MPP+), the MAO-B-dependent toxic metabolite of MPTP, or rotenone, two neurotoxins widely used to reproduce PD models in vivo and in vitro. The treatment with EtOH and βNF protected UD cells against MPP+ toxicity, whereas only βNF partially reverted the cytotoxic insult promoted by Rotenone. Moreover, the differentiated SH-SY5Y cells resulted to be less sensitive to the cytotoxic effects caused by the neurotoxins and consequently less responsive to the protection promoted by the CYPs induction. Furthermore, both βNF and EtOH treatments partially reverted the loss in mitochondria membrane potential, complex I impairment, as well as the increased ROS formation when UD were exposed to MPP+ or rotenone. Taken together, these results support the possible role of CYP isoforms in the neuroprotection against xenobiotic insult, especially as far as concerned the metabolic activity of CYP2D6 and CYP2E1. However, it would be interesting to investigate the induction dynamics of the CYPs here analysed and to understand the pathways triggered by βNF and EtOH leading to CYP induction. In conclusion, the impact that these isoforms have in the metabolism of other drugs, together with the present results, bring new insights on the role of brain CYPs in the effects of drugs and can drive future therapeutic approaches for ameliorating the therapy of neurological diseases.
Chiaino, E. (2022). CYP450 expression and regulation in SH-SY5Y cells. Possible role in neurotoxin-mediated injury.
Chiaino, Elda
File in questo prodotto:
File Dimensione Formato  
phd_unisi_085234.pdf

embargo fino al 14/06/2023

Descrizione: Tesi dottorato Chiaino
Tipologia: PDF editoriale
Licenza: PUBBLICO - Pubblico con Copyright
Dimensione 3.97 MB
Formato Adobe PDF
3.97 MB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11365/1211396