Cytotoxic and genotoxic evaluation of cotinine using human neuroblastoma cells (SH-SY5Y)

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.

Naringenin-Functionalized Gold Nanoparticles and Their Role in α-Synuclein Stabilization

“…The cytotoxicity measurement was performed using an MTT reduction assay where the amount of formazan converted in live cells can be quantified by measuring its absorbance at 570 nm. The assay evaluates mitochondrial activity and can be used to assess the overall cellular health . Treatment of cells with various doses (1 to 20 μM) of naringenin dissolved in 5% ethanol does not show any effect on cell viability (Figure ).…”

“…The assay evaluates mitochondrial activity and can be used to assess the overall cellular health. 69 Treatment of cells with various doses (1 to 20 μM) of naringenin dissolved in 5% ethanol does not show any effect on cell viability (Figure 9). Similarly, NAR-AuNPs did not affect cell viability up to a concentration of 400 μg/mL.…”

“…The overall absorbance was taken at 570 nm using a microplate reader. Results were expressed as a percentage of the control after subtracting the medium blank …”

Increased oxidative stress and mitochondrial impairments associated with increased expression of TNF‐α and caspase‐3 in palmitic acid‐induced lipotoxicity in myoblasts

“…The electrophoresis of DNA was then conducted for 15 min at 25 V (0.90 V/cm) and 300 mA as described. [ 46 ] Next, the slides were placed in a 0.4 M Tris buffer solution (pH 7.5) to neutralize and then stained with propidium iodide (2.5 μg/ml). The slides were examined at 400x magnification using an inverted fluorescence microscope (Leica) equipped with a video CCD camera.…”

“…The alkaline comet assay was carried out as described. [45,46] L6 myoblast cell suspension (20 μl) was dissolved in low melting agarose (80 μl) after the treatment and arranged in microscope slides. The microscopic slides were pre-coated with a layer of 1% normal melting point agarose.…”