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Mitogenic and functional responses by nicotine and hydrogen peroxide in AR42J cells: a comparative study.

Walker A, Udupa KB, Chowdhury P - Tob Induc Dis (2008)

Bottom Line: Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells.CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2.These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. PChowdhury@uams.edu.

ABSTRACT
The aim of the current study was to investigate the oxidative effects of nicotine by examining the mitogenic and functional responses in AR42J cells. As a control and for comparison, hydrogen peroxide (H2O2) was used as a source of known oxidative biomarker. Responses were examined by determining cell proliferation through the activation of ERK signaling, basal and CCK-stimulated cell function and measuring lipid peroxidation. AR42J cells have been exposed to either a non-cytotoxic dose of 20 muM H2O2 for 15 min or to 100 muM of nicotine for 3 min respectively. Nicotine and H2O2 at these dose and time intervals produced similar levels of malondialdyde (MDA) production and p-ERK1/2 activation. Immunofluorescence studies employing specific antibody to p-ERK1/2 confirmed the latter. Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells. CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2. These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production. The data suggests that nicotine-induced mitogenic response in AR42J cells closely identifies the response induced by an oxidative biomarker.

No MeSH data available.


Measurement of cytotoxic effect of H2O2. The cells were exposed to H2O2 in the dose range of 10–100 μM. A BioVision LDH-Cytotoxiciy Assay Kit (Mountain View, CA) was used, and the absorbance was measured after 24 hr incubation in 96-well plates at a wavelength of 495 nm. The percentage cytotoxicity was calculated as the ratio of absorptions of wells treated with H2O2 and untreated wells; N = 8, *, P < 0.05, significantly different from the uncxposed control.
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Figure 1: Measurement of cytotoxic effect of H2O2. The cells were exposed to H2O2 in the dose range of 10–100 μM. A BioVision LDH-Cytotoxiciy Assay Kit (Mountain View, CA) was used, and the absorbance was measured after 24 hr incubation in 96-well plates at a wavelength of 495 nm. The percentage cytotoxicity was calculated as the ratio of absorptions of wells treated with H2O2 and untreated wells; N = 8, *, P < 0.05, significantly different from the uncxposed control.

Mentions: In order to determine the cytotoxic dose of H2O2 on AR42J cells, cells were exposed for 24 h to a graded dose of H2O2. The percent cytotoxicity as determined by LDH release is shown in Figure 1. In untreated control cells the percent of LDH release was 5.4 ± 1.1% while cells exposed to 20 μM H2O2 it was 6.7 ± 3.9%. This value was not significantly different from the control. As the concentration of H2O2 increased beyond 20 μM, the percentage of cytotoxic cells was increased. With 100 μM H2O2, it was 28.0 ± 0.3%. This is not surprising since it has been reported that EL-4 murine lymphoma cells exposed to H2O2 doses beyond 20 μM, increased number of cytotoxic cells was found by Zhou et al [20]. Thus we have used this non-cytotoxic concentration of 20 μM of H2O2 in all the subsequent studies.


Mitogenic and functional responses by nicotine and hydrogen peroxide in AR42J cells: a comparative study.

Walker A, Udupa KB, Chowdhury P - Tob Induc Dis (2008)

Measurement of cytotoxic effect of H2O2. The cells were exposed to H2O2 in the dose range of 10–100 μM. A BioVision LDH-Cytotoxiciy Assay Kit (Mountain View, CA) was used, and the absorbance was measured after 24 hr incubation in 96-well plates at a wavelength of 495 nm. The percentage cytotoxicity was calculated as the ratio of absorptions of wells treated with H2O2 and untreated wells; N = 8, *, P < 0.05, significantly different from the uncxposed control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2556029&req=5

Figure 1: Measurement of cytotoxic effect of H2O2. The cells were exposed to H2O2 in the dose range of 10–100 μM. A BioVision LDH-Cytotoxiciy Assay Kit (Mountain View, CA) was used, and the absorbance was measured after 24 hr incubation in 96-well plates at a wavelength of 495 nm. The percentage cytotoxicity was calculated as the ratio of absorptions of wells treated with H2O2 and untreated wells; N = 8, *, P < 0.05, significantly different from the uncxposed control.
Mentions: In order to determine the cytotoxic dose of H2O2 on AR42J cells, cells were exposed for 24 h to a graded dose of H2O2. The percent cytotoxicity as determined by LDH release is shown in Figure 1. In untreated control cells the percent of LDH release was 5.4 ± 1.1% while cells exposed to 20 μM H2O2 it was 6.7 ± 3.9%. This value was not significantly different from the control. As the concentration of H2O2 increased beyond 20 μM, the percentage of cytotoxic cells was increased. With 100 μM H2O2, it was 28.0 ± 0.3%. This is not surprising since it has been reported that EL-4 murine lymphoma cells exposed to H2O2 doses beyond 20 μM, increased number of cytotoxic cells was found by Zhou et al [20]. Thus we have used this non-cytotoxic concentration of 20 μM of H2O2 in all the subsequent studies.

Bottom Line: Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells.CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2.These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. PChowdhury@uams.edu.

ABSTRACT
The aim of the current study was to investigate the oxidative effects of nicotine by examining the mitogenic and functional responses in AR42J cells. As a control and for comparison, hydrogen peroxide (H2O2) was used as a source of known oxidative biomarker. Responses were examined by determining cell proliferation through the activation of ERK signaling, basal and CCK-stimulated cell function and measuring lipid peroxidation. AR42J cells have been exposed to either a non-cytotoxic dose of 20 muM H2O2 for 15 min or to 100 muM of nicotine for 3 min respectively. Nicotine and H2O2 at these dose and time intervals produced similar levels of malondialdyde (MDA) production and p-ERK1/2 activation. Immunofluorescence studies employing specific antibody to p-ERK1/2 confirmed the latter. Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells. CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2. These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production. The data suggests that nicotine-induced mitogenic response in AR42J cells closely identifies the response induced by an oxidative biomarker.

No MeSH data available.