Tuberc Respir Dis.  2007 Mar;62(3):197-202. 10.4046/trd.2007.62.3.197.

Effects of Nicotine, Cotinine and Benzopyrene as Smoke Components on the Expression of Antioxidants in Human Bronchial Epithelial Cells

• Kim YS
• Lee JH
• Kim SH
• Kim TH
• Sohn JW
• Yoon HJ
• Park SS
• Shin DH

• KMID: 1518464
• DOI: http://doi.org/10.4046/trd.2007.62.3.197

Abstract

BACKGROUND: Cigarette smoking is an important risk factor for chronic bronchitis and COPD. Airway epithelial cells exposed to cigarette smoke components such as nicotine, cotinine and benzopyrene can generate reactive oxygen species (ROS) and be subject to oxidative stress. This oxidative stress can induce the inflammatory response in the lung by the oxidant itself or by the release of proinflammatory cytokines. It has been reported that nicotine stimulates ROS, which are associated with NF-kappaB.
METHODS
Beas2B cells were treated with nicotine, cotinine and benzopyrene. RT PCR was used to measure the expression of several antioxidant factors using the total RNA from the Beas2B cells. The level of superoxide dismutase(CuZnSOD), thioredoxin, glutathione reductase expression was examined.
RESULTS
0.5 to 4 hours after the benzopyrene, nicotine and cotinine theatments, the level of thioredoxin and glutathione reductase expression decreased. Longer exposure to these compounds for 24 to 72 hours inhibited the expression of most of these antioxidant factors.
CONCLUSION
During exposure to smoke compounds, thioredoxin and glutathione reductase are the key antioxidant factors induced sensitively between 0.5 and 4 hours but the levels these antioxidants decrease between 24 hour and 72hours.

Keyword

Beas2B cell; Benzopyrene; Nicotine; Cotinine; Antioxidants

MeSH Terms

Antioxidants*
Bronchitis, Chronic
Cotinine*
Cytokines
Epithelial Cells*
Glutathione Reductase
Humans*
Lung
NF-kappa B
Nicotine*
Oxidative Stress
Polymerase Chain Reaction
Pulmonary Disease, Chronic Obstructive
Reactive Oxygen Species
Risk Factors
RNA
Smoke*
Smoking
Superoxides
Thioredoxins
Tobacco Products
Antioxidants
Cotinine
Cytokines
Glutathione Reductase
NF-kappa B
Nicotine
RNA
Reactive Oxygen Species
Smoke
Superoxides
Thioredoxins

Figure

• 

  Figure 1 Expression of antioxidants of Beas2B cell treated with smoke components(benzopyrene, nicotine, cotinine) at 0.5, 1, 4, 24, 48, 72hrs post-treatment time. The expression level of CuZnSOD(A), thioredoxin(B), and glutathione reductase(C) as an intracellular antioxidants was analyzed after treatment of Beas2B cell with benzopyrene, nicotine, or cotinine. Those levels were determined as the PCR band intensities measured by Quantity One program(Bio-Rad).The fold change was calculated as the expression level of an antioxidant from Beas2B cell treated with a smoke component divided by the level of negative control(no treatment of smoke components) at individual post-treatment time.

• 

  Figure 2 RT PCR of antioxidants of Beas2B cell treated with smoke components. PCR results of cDNA from Beas2B cell with no treatment(A. CONTROL) or with 150nM BENZO(a)PYRENE(B. BENZOPYRENE) or with 10mM NICOTINE(C. NICOTINE) or with 10mM COTININE(D. COTININE) were taken at 0.5, 1, 4, 24, 48, 72 hrs post-treatment. CuZnSOD1 - Cu/ZnSuperoxide Dismutase(217bp) TR2 - Thioredoxin(274bp) GR3 - Glutathione Reductase(433bp) GAPDH(307bp) ✶Size marker(GeneRuler DNA Ladder Mix, Fermentas)

• 

  Figure 3 Effect of smoke components on NFkB reporter activity of respiratory tract cells The regulatory mechanism of antioxidant by smoke component was investigated concerned with NF-kB activation. NFkB reporter DNA(Clontech ,USA) was transiently transfected into Beas2B cell with Lipofectin Reagent® (Invitrogen, USA) and Firefly luciferase assay was done. Cotinine and benzopyrene as smoke components stimulated the activity of NFkB reporter of Beas2B cell lines.

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