Abstract
Cytochrome P450 (CYP) isozymes vary their expression depending on the brain area, the cell type, and the presence of drugs. Some isoforms are involved in detoxification and/or toxic activation of xenobiotics in central nervous system. However, their role in brain metabolism and neurodegeneration is still a subject of debate. We have studied the inducibility of CYP isozymes in human neuroblastoma SH-SY5Y cells, treated with β-naphtoflavone (β-NF) or ethanol (EtOH) as inducers, by qRT-PCR, Western blot (WB), and metabolic activity assays. Immunohistochemistry was used to localize the isoforms in mitochondria and/or endoplasmic reticulum (ER). Tetrazolium (MTT) assay was performed to study the role of CYPs during methylphenyl pyridine (MPP+) exposure. EtOH increased mRNA and protein levels of CYP2D6 by 73% and 60% respectively. Both β-NF and EtOH increased CYP2E1 mRNA (4- and 1.4-fold, respectively) and protein levels (64% both). The 7-ethoxycoumarin O-deethylation and dextromethorphan O-demethylation was greater in treatment samples than in controls. Furthermore, both treatments increased by 22% and 18%, respectively, the cell viability in MPP+-treated cells. Finally, CYP2D6 localized at mitochondria and ER. These data indicate that CYP is inducible in SH-SY5Y cells and underline this in vitro system for studying the role of CYPs in neurodegeneration.
Highlights
Cytochrome P450 (CYP) is a superfamily of isozymes involved in the metabolism of xenobiotics in liver and other extra-hepatic tissues
Our results demonstrate that CYP2D6 and 2E1 can be increased in terms of mRNA, proteins, and activities by incubating undifferentiated SH-SY5Y cells with two well-known inducer compounds in the liver, β-NF and EtOH. β-NF is known to induce CYP isoforms belonging to the family 1A and 1B, while EtOH induces the expression of CYP2E1 isoform
CYP1A1 was unexpectedly not induced by β-NF. These results indicate that the regulation pathways of CYPs in SH-SY5Y cells are different compared to those observed in liver in vivo and in vitro conditions
Summary
Cytochrome P450 (CYP) is a superfamily of isozymes involved in the metabolism of xenobiotics in liver and other extra-hepatic tissues. TThhee rreessuullttss sshhoowweedd tthhaatt tthhee mmaaxxiimmuumm ccoonncceennttrraattiioonn tthhaatt hhaadd nnoo eeffffeecctt oonn SSHH--SSYY55YY cceellll vviiaabbiilliittyy aafftteerr 4488 hh ooff iinnccuubbaattiioonn wweerree 44 μμMM ffoorr ββ--NNFF aanndd 110000 mmMM ffoorr EEttOOHH ((FFiigguurree SS11)). IInn uunnddiiffffeerreennttiiaatteedd cceellllss,, tthhee mmRRNNAA lleevveellss ooff CCYYPP22DD66 wweerree nnoott ssiiggnniifificcaannttllyy aaffffeecctteedd bbyy ββ--NNFF ttrreeaattmmeenntt,, yyeett EEttOOHH pprroommootteedd aa ssiiggnniifificcaattiivvee 11..77--ffoolldd iinnccrreeaassee ((FFiigguurree 11aa)). This can be explained by the fact that dextromethorphan is only metabolized by CYP2D6, unlike 7-ethoxycoumarin, and its low concentration in our cellular preparations, even in the case of induction, is not sufficient to increase the formation of dextrorphane in a statistically significant manner Another question to address is the intracellular expression of these isoforms in SH-SY5Y cells.
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