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Updated: Dec 20, 2025
Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line
Published on: February 17, 2016
Cytotoxic and genotoxic evaluation of cotinine using human neuroblastoma cells (SH-SY5Y)
Daiana Dalberto1, Caroline Cardoso Nicolau1, Ana Leticia Hilario Garcia1,2
1Universidade Luterana do Brasil (ULBRA), Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde - PPGBioSaúde, Laboratório de Toxicologia Genética, Canoas, RS, Brazil.
Abstract:
Cotinine is the main metabolite of nicotine, which is metabolized in the liver through a cytochrome P450 enzyme. Different studies point to genetic instability caused by nicotine, such as single and double DNA strand breaks and micronuclei formation, but little is known about the effect of cotinine. Therefore, the present in vitro study assessed the effects of cotinine on cell viability and DNA damage in SH-SY5Y neuroblastoma cells, as well as genotoxicity related to oxidative stress mechanisms. Comparisons with nicotine were also performed. An alkaline comet assay modified by repair endonucleases (FPG, OGG1, and Endo III) was used to detect oxidized nucleobases. SH-SY5Y neuronal cells were cultured under standard conditions and exposed for 3 h to different concentrations of cotinine and nicotine. Cytotoxicity was observed at higher doses of cotinine and nicotine in the MTT assay. In the trypan blue assay, cells showed viability above 80% for both compounds. Alkaline comet assay results demonstrated a significant increase in damage index and frequency for cells treated with cotinine and nicotine, presenting genotoxicity. The results of the enzyme-modified comet assay suggest a DNA oxidative damage induced by nicotine. Unlike other studies, our results demonstrated genotoxicity induced by both cotinine and nicotine. The similar effects observed for these two pyridine alkaloids may be due to the similarity of their structures.
Insights
Cotinine and nicotine cause DNA damage and genotoxicity in neuronal cells. This study reveals cotinine
Area of Science:
- Neuroscience
- Toxicology
- Genetics
Background:
- Nicotine is known to cause genetic instability, including DNA strand breaks and micronuclei formation.
- The genotoxic effects of cotinine, nicotine's primary metabolite, are not well understood.
- Understanding cotinine's impact is crucial given its prevalence as a nicotine breakdown product.
Purpose of the Study:
- To evaluate the effects of cotinine on cell viability and DNA damage in SH-SY5Y neuroblastoma cells.
- To investigate cotinine-induced genotoxicity and its relation to oxidative stress mechanisms.
- To compare the genotoxic effects of cotinine with those of nicotine.
Main Methods:
- Utilized an in vitro model with SH-SY5Y neuroblastoma cells.
- Assessed cell viability using MTT and trypan blue assays.
- Employed an alkaline comet assay, modified with repair endonucleases (FPG, OGG1, Endo III), to detect oxidized nucleobases and DNA damage.
Main Results:
- Both cotinine and nicotine exhibited cytotoxicity at higher concentrations (MTT assay).
- Cell viability remained above 80% for both compounds across tested concentrations (trypan blue assay).
- Significant increases in DNA damage index and frequency were observed for both cotinine and nicotine treatments, indicating genotoxicity.
- Enzyme-modified comet assays suggested nicotine induces DNA oxidative damage.
- This study demonstrated genotoxicity for both cotinine and nicotine, contrary to some previous findings.
Conclusions:
- Cotinine, like nicotine, induces genotoxicity and DNA damage in neuronal cells.
- The structural similarity between cotinine and nicotine may explain their comparable effects.
- Further research is warranted to elucidate the precise mechanisms of cotinine-induced genotoxicity and oxidative stress.
