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Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

Naik P, Fofaria N, Prasad S, Sajja RK, Weksler B, Couraud PO, Romero IA, Cucullo L - BMC Neurosci (2014)

Bottom Line: Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, "reduced-exposure" brand) and ultralow nicotine products.In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products.In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, School of Pharmacy, 1300 S, Coulter Street, Amarillo TX 79106, USA. luca.cucullo@ttuhsc.edu.

ABSTRACT

Background: Both active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood-brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer's disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model.

Results: Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, "reduced-exposure" brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1.

Conclusions: In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content.

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HPLC and viability studies to select the CSE concentration for the study. A) HPLC analysis to determine nicotine concentration in CSEshowed that 5% CSE had nicotine concentration comparable to the physiological concentration in a chronic smoker (100 ng/ml) n = 10. (B) Cell viability studies following increasing concentration of nicotine at 24 and 48 h and (C) 5% diluted CSE from ultralow, 1R5F (equivalent to ultralight cigarettes), 3R4F (equivalent to full flavor cigarettes), ultralow nicotine and tobacco free (nicotine free - NF) using MTT assay. Note that 5% CSE from all tested brand but NF did not cause a statistically significant decrease in cell viability. n = 3 individual experiments.
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Figure 1: HPLC and viability studies to select the CSE concentration for the study. A) HPLC analysis to determine nicotine concentration in CSEshowed that 5% CSE had nicotine concentration comparable to the physiological concentration in a chronic smoker (100 ng/ml) n = 10. (B) Cell viability studies following increasing concentration of nicotine at 24 and 48 h and (C) 5% diluted CSE from ultralow, 1R5F (equivalent to ultralight cigarettes), 3R4F (equivalent to full flavor cigarettes), ultralow nicotine and tobacco free (nicotine free - NF) using MTT assay. Note that 5% CSE from all tested brand but NF did not cause a statistically significant decrease in cell viability. n = 3 individual experiments.

Mentions: HPLC studies were performed to determine the dilution factor for freshly prepared 3R4F cigarette-derived CSE stock solution necessary to achieve CSE exposure yielding 100 ng/ml of nicotine (Figure 1A). This nicotine concentration was chosen to model the plasma levels seen in human smokers [27-29]. 3R4F cigarette was used as a reference to calculate the dilution factor for the CSE stock which was then uniformly applied to all the test cigarettes. As shown in Figure 1B, 100 ng/ml of nicotine did not affect the cell viability at 24 and 48 h exposure. Cytotoxic effects of nicotine exposure were observed at higher concentrations (10 and 100 μg/ml/24 h; 1, 10, 100 μg/ml/48 h). Note also that 24 h exposure to 5% diluted CSE from test cigarettes did not affect endothelial viability with the exception of NF-derived extracts (see Figure 1C). A small yet significant decrease in cell viability was observed in response to NF-derived CSE exposure, as compared to controls (CSE-free PBS or 100 ng/ml nicotine treatment).


Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

Naik P, Fofaria N, Prasad S, Sajja RK, Weksler B, Couraud PO, Romero IA, Cucullo L - BMC Neurosci (2014)

HPLC and viability studies to select the CSE concentration for the study. A) HPLC analysis to determine nicotine concentration in CSEshowed that 5% CSE had nicotine concentration comparable to the physiological concentration in a chronic smoker (100 ng/ml) n = 10. (B) Cell viability studies following increasing concentration of nicotine at 24 and 48 h and (C) 5% diluted CSE from ultralow, 1R5F (equivalent to ultralight cigarettes), 3R4F (equivalent to full flavor cigarettes), ultralow nicotine and tobacco free (nicotine free - NF) using MTT assay. Note that 5% CSE from all tested brand but NF did not cause a statistically significant decrease in cell viability. n = 3 individual experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4020608&req=5

Figure 1: HPLC and viability studies to select the CSE concentration for the study. A) HPLC analysis to determine nicotine concentration in CSEshowed that 5% CSE had nicotine concentration comparable to the physiological concentration in a chronic smoker (100 ng/ml) n = 10. (B) Cell viability studies following increasing concentration of nicotine at 24 and 48 h and (C) 5% diluted CSE from ultralow, 1R5F (equivalent to ultralight cigarettes), 3R4F (equivalent to full flavor cigarettes), ultralow nicotine and tobacco free (nicotine free - NF) using MTT assay. Note that 5% CSE from all tested brand but NF did not cause a statistically significant decrease in cell viability. n = 3 individual experiments.
Mentions: HPLC studies were performed to determine the dilution factor for freshly prepared 3R4F cigarette-derived CSE stock solution necessary to achieve CSE exposure yielding 100 ng/ml of nicotine (Figure 1A). This nicotine concentration was chosen to model the plasma levels seen in human smokers [27-29]. 3R4F cigarette was used as a reference to calculate the dilution factor for the CSE stock which was then uniformly applied to all the test cigarettes. As shown in Figure 1B, 100 ng/ml of nicotine did not affect the cell viability at 24 and 48 h exposure. Cytotoxic effects of nicotine exposure were observed at higher concentrations (10 and 100 μg/ml/24 h; 1, 10, 100 μg/ml/48 h). Note also that 24 h exposure to 5% diluted CSE from test cigarettes did not affect endothelial viability with the exception of NF-derived extracts (see Figure 1C). A small yet significant decrease in cell viability was observed in response to NF-derived CSE exposure, as compared to controls (CSE-free PBS or 100 ng/ml nicotine treatment).

Bottom Line: Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, "reduced-exposure" brand) and ultralow nicotine products.In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products.In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, School of Pharmacy, 1300 S, Coulter Street, Amarillo TX 79106, USA. luca.cucullo@ttuhsc.edu.

ABSTRACT

Background: Both active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood-brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer's disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model.

Results: Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, "reduced-exposure" brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1.

Conclusions: In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content.

Show MeSH
Related in: MedlinePlus