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. 2014 Apr 23:15:51.
doi: 10.1186/1471-2202-15-51.

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?

Pooja Naik, Neel Fofaria, Shikha Prasad, Ravi K Sajja, Babette Weksler, Pierre-Olivier Couraud, Ignacio A Romero, Luca Cucullo  1
Affiliations

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?

Pooja Naik et al. BMC Neurosci. .

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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Figures

Figure 1
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.
Figure 2
Figure 2
Nitrate/Nitrite content profiling of CSE from tested tobacco products. (A) Nitrate/nitrite content increased proportionally to the amount of tar in the cigarettes where statistically significant higher nitrate was found in ultralow nicotine than ultralight cigarette (P < 0.001, n = 10 biological replicates). Nicotine free cigarettes which were non-tobacco based did not have significant nitrate/nitrite content. Regression analysis of Nitrate/Nitrite correlates with tar (B) but not nicotine content unless ultralow nicotine cigarettes are removed from the analytical pool (C).
Figure 3
Figure 3
Hydrogen Peroxide content profiling of CSE from tested tobacco products. (A1) H2O2 content increased proportionally to the amount of tar in the cigarettes where statistically significant higher H2O2 was found in full flavor, ultralow nicotine and nicotine free cigarettes than light cigarette (P < 0.001), (n = 10 CSE preparations). (A2) Immunofluorescence analysis of oxidative stress in ECs (HCMEC/D3 cell line) caused by CSE exposure from 1R5F, 3R4F, ultralow nicotine and NF cigarettes versus controls: Note that most significant oxidative responses were observed in EC cultures exposed to CSE treatment (24 h) derived from 3R4F, ultralow nicotine and NF cigarette (n = 3 biological replicates). Regression analysis of H2O2 correlates with tar (B) but not nicotine content unless ultralow nicotine and NF cigarettes are removed from the analytical pool (C).
Figure 4
Figure 4
Effect of CSE exposure (24 h) on endothelial expression and distribution of ZO-1, Occludin and actin filaments. (A) Down-regulation of ZO-1 expression and disruption and cell-cell junctions were progressively more significant following exposure to 3R4F, NF and ultralow nicotine CSEs. Down-regulation and disruption at cell-cell contacts of occludin were also observed (B). Immunofluorescence analyses were confirmed by WB of corresponding membrane fractions (C). Loss of BBB integrity was further assessed by permeability measurements to dextran molecules ranging from 3 to 70 kDa (D) n = 3 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001 compared to controls.
Figure 5
Figure 5
Effect of CSE exposure (24 h) on endothelial expression and distribution of VE-cadherin and Claudin-5. (A) Immunofluorescence analysis of BBB endothelial cultures revealed a significant up-regulation of VE-cadherin (at cell-cell junctions) and claudin-5 expression following exposure to 3R4F, NF and ultralow nicotine CSEs. (B) Immunofluorescence analyses were confirmed by WB of corresponding membrane fractions. n = 3 biological replicates, *p < 0.05, **p < 0.01 compared to controls.
Figure 6
Figure 6
Immunofluorescence analysis of BBB endothelial expression of VCAM-1 and E-selectin (A) and PECAM1 (B), following exposure to CSEs from 1R5F, 3R4F, NF and ultralow nicotine cigarettes. Immunofluorescence analysis of PECAM1 was confirmed by WB of corresponding membrane fractions. (C) Release of proinflammatory cytokines IL-6 was up-regulated in endothelial cultures exposed to NF and ultralow nicotine CSE while MMP-2 levels were increased by CSE from 3R4F and NF but not ultralow nicotine. n = 3 biological replicates, *p < 0.05, **p < 0.01, ****p < 0.0001 compared to controls.
Figure 7
Figure 7
Smoke Preparation according to ISO/FTC protocol. Concentrated Smoke solution was prepared from each cigarette using CSM-SCSM Cigarette Smoking Machine according to ISO/FTC determination parameters. These require a puff volume of 35 ml with duration of 2 s at interval of 60s.
Figure 8
Figure 8
Tar and Nicotine content of the main tobacco products used in the study. The following table states the tar and nicotine composition of the various cigarettes used in the study. (+ 1R5F and 3R4F are the names of these cigarettes provided by University of Kentucky for research purposes).
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