Nicotine induces oxidative stress and activates nuclear transcription factor kappa B in rat mesencephalic cells

Abstract

Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants. Toxicity exhibited by cigarette smoke may be due to combined action of these compounds inducing many cellular processes mediated through reactive oxygen species (ROS). Major player probably nicotine as it is present in tobacco, in higher concentrations. The compounds that induce intracellular oxidative stress recognized as the important agents involved in the damage of biological molecules. Experiments using animal and cell culture model systems suggested that moderately higher concentrations of some forms of ROS like NO and H(2)O(2) can act as signal transducing agents. Nuclear transcription factor kappaB (NF-kappaB) an inducible transcription factor detected in neurons found to be involved in many biological processes such as inflammation, innate immunity, development, apoptosis, and antiapoptosis. Our present study demonstrates that nicotine induces ROS levels in a dose dependent manner in rat mesencephalic cells. Electro mobility shift analysis showed that nicotine activates inducible NF-kappaB by binding to consensus sequence of DNA. Nicotine added to cell culture stimulates the degradation of IkappaB-alpha subunit in 2 h. Further activation of c-Jun terminal kinase indicates that nicotine induces oxidative stress leading to activation of stress dependent NF-kappaB pathway in mesencephalic cells.

Fig. 1

Effect of Nicotine on ROS levels in rat Mesencephalic Cells. Cells were treated with nicotine at concentrations 0.1, 1.0 and 10.0 μM and incubated for 3 h. Fluorescence was measured at the end of 3 h. and expressed as percent of control florescence mean ± SD of 8 wells (n = 8) and the figure is a representative from three experiments performed independently. The * indicates significantly different from control samples.

Fig. 2

Time course of induction of ROS by Nicotine in rat mesencephalic cells. Cells were incubated with 10 μM of nicotine. Fluorescence was measured at different time intervals as described in materials and methods. The values expressed as percent of control florescence mean ± SD of 8 wells (n = 8) and the figure is a representative from three experiments performed independently.

Fig. 3

Effect of nicotine on NF-κB DNA binding activity in mesencephalic cells (dose). Nuclear proteins (20 μg) were analyzed on 7.5% native PAGE to detect NF-κB by electrophoretic mobility shift assay. The data shown is a representative of three independent experiments performed on extracts obtained from control and different concentrations of nicotine (0.1, 1.0, 5.0, 10 μM) treated mesencephalic cells.

Fig. 4

Effect of nicotine on NF-κB DNA binding activity in mesencephalic cells (time). Nuclear proteins (20 μg) were analyzed on 7.5% native PAGE to detect NF-κB by electrophoretic mobility shift assay. The data shown is a representative of three independent experiments performed on extracts obtained from control and 1 μM nicotine treated mesencephalic cells for different time periods.

Fig. 5

Effect of nicotine on IκB-α degeradation. Mesencephalic cells were treated with or without nicotine (1 μM) for different time intervals as described in materials and methods. Cells were washed with PBS and cell extracts were made in PBS by homogenization. Equal quantity of protein (75 μg) was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nylon membranes and probed with IκB-α antibodies washed thrice with PBS and incubated with second antibody horse radish peroxidase and bands were analyzed by chemiluminiscence kit.

Fig. 6

Effect of nicotine on JNK activation. Cell extracts from control and nicotine treated mesencephalic cells were prepared as described in materials and methods and 300 μg of total protein extract were immuno-precipitated with anti-JNK (0.3 μg/sample) antibody, and the kinase reaction was performed using ³²P-labeled ATP and GST-Jun as substrate protein. The radiolabeled GST-Jun was detected in 9% SDS-PAGE. The level of JNK was detected using 50 μg of same extract proteins and analyzed in 10% SDS-PAGE by Western blot.