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Effect of full flavor and denicotinized cigarettes exposure on the brain microvascular endothelium: a microarray-based gene expression study using a human immortalized BBB endothelial cell line.

Naik P, Sajja RK, Prasad S, Cucullo L - BMC Neurosci (2015)

Bottom Line: Several detoxification and anti-oxidant genes including downstream products were also activated including NAD(P)H dehydrogenase quinone 1 (NQO-1), heme oxygenase-1 (HMOX-1), catalytic and modifier subunits of glutamate-cysteine ligase (GCL), solute carrier-SLC7A11).Gene expression levels of cytochrome P450s (CYP2S1 and CYP51A1) and efflux transporters P-glycoprotein (P-gp) and multi-drug resistance protein-4 (MRP4) were also enhanced.Increase of P-gp functional activity and depletion of GSH were also observed.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: Tobacco smoke (TS) toxicity to the brain microvasculature is still an understudied area till date. NF-E2 related factor (Nrf2) is a key transcription factor responsible for activating the antioxidant response element (ARE) genes following an oxidative insult. Till date, several studies targeting the blood brain barrier (BBB) have shown some protective role of Nrf2 in ischemia-reperfusion (IR) injury, however, its functional role in chronic smokers subjected to a life-long oxidative stress has never been addressed. This is of crucial importance since smokers have a much higher risk for cerebrovascular stroke and tobacco smoke exposure has been clearly shown to enhance BBB damage following an ischemia/reperfusion injury. Thus, the goal of our study was to investigate the defense pathways activated at the BBB endothelial level by TS exposure. Specifically we focused on Nrf2 and nuclear factor kappa-light-chain-enhancer of activated B signaling response (NF-κβ) as the central protective mechanisms related to oxidative insult.

Results: With the exception of Nicotine, both full flavor (3R4F) and decotinized (ULN) cigarettes activated Nrf2 and NFκβ pathways in hCMEC/D3 endothelial cells. Several detoxification and anti-oxidant genes including downstream products were also activated including NAD(P)H dehydrogenase quinone 1 (NQO-1), heme oxygenase-1 (HMOX-1), catalytic and modifier subunits of glutamate-cysteine ligase (GCL), solute carrier-SLC7A11). Gene expression levels of cytochrome P450s (CYP2S1 and CYP51A1) and efflux transporters P-glycoprotein (P-gp) and multi-drug resistance protein-4 (MRP4) were also enhanced. Increase of P-gp functional activity and depletion of GSH were also observed. Strikingly, toxicity of denicotinized ("reduced exposure") cigarettes was equivalent to 3R4F (or worse).

Conclusions: This study provides a detailed analysis of Nrf2-related cytoprotective mechanisms activated in response to 3R4F and ULN-derived TS exposure correlating the results with their oxidative and inflammatory potential. Toxicants present in soluble cigarette smoke extracts (CSE) and not nicotine seem to be the primary determinant of vascular toxicity. In this respect our results from this and previous studies suggest that chronic TS exposure can overcome Nrf2 and NFκB-p65 dependent cytoprotective mechanisms of the brain microvascular endothelium possibly leading to BBB impairment and loss of BBB integrity.

No MeSH data available.


Related in: MedlinePlus

CSE exposure of various TS products induces inflammatory stress in BBB ECs. a1 mRNA expression of NFκB-p65 in hCMEC/D3 cells following exposure to nicotine, CSE from both tobacco products; a2 western blot analysis of cytosolic and nuclear fractions of NFκB-p65 showed increased protein expression and nuclear localization following CSEs; representative western blots were shown with actin as a loading control; a3 the translocation status could be better understood via ratio of cytosolic versus nuclear NFκB-p65 plotted in terms of percent control; b release of chemokines such as IL-8 and MCP-1 from hCMEC/D3 cell cultures as determined by ELISA; c VEGF release was up-regulated in endothelial cultures exposed to full flavor and ULN CSE but not nicotine. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control. n = 6 biological replicates/condition.
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Fig7: CSE exposure of various TS products induces inflammatory stress in BBB ECs. a1 mRNA expression of NFκB-p65 in hCMEC/D3 cells following exposure to nicotine, CSE from both tobacco products; a2 western blot analysis of cytosolic and nuclear fractions of NFκB-p65 showed increased protein expression and nuclear localization following CSEs; representative western blots were shown with actin as a loading control; a3 the translocation status could be better understood via ratio of cytosolic versus nuclear NFκB-p65 plotted in terms of percent control; b release of chemokines such as IL-8 and MCP-1 from hCMEC/D3 cell cultures as determined by ELISA; c VEGF release was up-regulated in endothelial cultures exposed to full flavor and ULN CSE but not nicotine. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control. n = 6 biological replicates/condition.

Mentions: We next determined the impact of TS and nicotine on the p65 subunit of NFκB (NFκB-p65) gene expression, a transcriptional factor and potential activator of oxidative and inflammatory stress pathways [22]. As shown in Figure 7a1, the mRNA expression of NFκB-p65 was markedly elevated by CSE exposure of both products with ULN producing a stronger response in 3R4F. We also observed a significant increase in the activation and nuclear translocation of NFκB-p65 following exposure to either 3R4F or ULN smoke extracts (Figure 7a2). As shown in Figure 7a3, the cytoplasm/nuclear ratio of NFκB-p65 in BBB endothelial cultures exposed to 3R4F or ULN smoke extracts was only 50% of that measured in controls. By contrast, nicotine exposure alone did altered neither the mRNA expression nor the nuclear translocation of NFκB-p65 (Figure 7a1–a3).Figure 7


Effect of full flavor and denicotinized cigarettes exposure on the brain microvascular endothelium: a microarray-based gene expression study using a human immortalized BBB endothelial cell line.

Naik P, Sajja RK, Prasad S, Cucullo L - BMC Neurosci (2015)

CSE exposure of various TS products induces inflammatory stress in BBB ECs. a1 mRNA expression of NFκB-p65 in hCMEC/D3 cells following exposure to nicotine, CSE from both tobacco products; a2 western blot analysis of cytosolic and nuclear fractions of NFκB-p65 showed increased protein expression and nuclear localization following CSEs; representative western blots were shown with actin as a loading control; a3 the translocation status could be better understood via ratio of cytosolic versus nuclear NFκB-p65 plotted in terms of percent control; b release of chemokines such as IL-8 and MCP-1 from hCMEC/D3 cell cultures as determined by ELISA; c VEGF release was up-regulated in endothelial cultures exposed to full flavor and ULN CSE but not nicotine. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control. n = 6 biological replicates/condition.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477310&req=5

Fig7: CSE exposure of various TS products induces inflammatory stress in BBB ECs. a1 mRNA expression of NFκB-p65 in hCMEC/D3 cells following exposure to nicotine, CSE from both tobacco products; a2 western blot analysis of cytosolic and nuclear fractions of NFκB-p65 showed increased protein expression and nuclear localization following CSEs; representative western blots were shown with actin as a loading control; a3 the translocation status could be better understood via ratio of cytosolic versus nuclear NFκB-p65 plotted in terms of percent control; b release of chemokines such as IL-8 and MCP-1 from hCMEC/D3 cell cultures as determined by ELISA; c VEGF release was up-regulated in endothelial cultures exposed to full flavor and ULN CSE but not nicotine. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control. n = 6 biological replicates/condition.
Mentions: We next determined the impact of TS and nicotine on the p65 subunit of NFκB (NFκB-p65) gene expression, a transcriptional factor and potential activator of oxidative and inflammatory stress pathways [22]. As shown in Figure 7a1, the mRNA expression of NFκB-p65 was markedly elevated by CSE exposure of both products with ULN producing a stronger response in 3R4F. We also observed a significant increase in the activation and nuclear translocation of NFκB-p65 following exposure to either 3R4F or ULN smoke extracts (Figure 7a2). As shown in Figure 7a3, the cytoplasm/nuclear ratio of NFκB-p65 in BBB endothelial cultures exposed to 3R4F or ULN smoke extracts was only 50% of that measured in controls. By contrast, nicotine exposure alone did altered neither the mRNA expression nor the nuclear translocation of NFκB-p65 (Figure 7a1–a3).Figure 7

Bottom Line: Several detoxification and anti-oxidant genes including downstream products were also activated including NAD(P)H dehydrogenase quinone 1 (NQO-1), heme oxygenase-1 (HMOX-1), catalytic and modifier subunits of glutamate-cysteine ligase (GCL), solute carrier-SLC7A11).Gene expression levels of cytochrome P450s (CYP2S1 and CYP51A1) and efflux transporters P-glycoprotein (P-gp) and multi-drug resistance protein-4 (MRP4) were also enhanced.Increase of P-gp functional activity and depletion of GSH were also observed.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: Tobacco smoke (TS) toxicity to the brain microvasculature is still an understudied area till date. NF-E2 related factor (Nrf2) is a key transcription factor responsible for activating the antioxidant response element (ARE) genes following an oxidative insult. Till date, several studies targeting the blood brain barrier (BBB) have shown some protective role of Nrf2 in ischemia-reperfusion (IR) injury, however, its functional role in chronic smokers subjected to a life-long oxidative stress has never been addressed. This is of crucial importance since smokers have a much higher risk for cerebrovascular stroke and tobacco smoke exposure has been clearly shown to enhance BBB damage following an ischemia/reperfusion injury. Thus, the goal of our study was to investigate the defense pathways activated at the BBB endothelial level by TS exposure. Specifically we focused on Nrf2 and nuclear factor kappa-light-chain-enhancer of activated B signaling response (NF-κβ) as the central protective mechanisms related to oxidative insult.

Results: With the exception of Nicotine, both full flavor (3R4F) and decotinized (ULN) cigarettes activated Nrf2 and NFκβ pathways in hCMEC/D3 endothelial cells. Several detoxification and anti-oxidant genes including downstream products were also activated including NAD(P)H dehydrogenase quinone 1 (NQO-1), heme oxygenase-1 (HMOX-1), catalytic and modifier subunits of glutamate-cysteine ligase (GCL), solute carrier-SLC7A11). Gene expression levels of cytochrome P450s (CYP2S1 and CYP51A1) and efflux transporters P-glycoprotein (P-gp) and multi-drug resistance protein-4 (MRP4) were also enhanced. Increase of P-gp functional activity and depletion of GSH were also observed. Strikingly, toxicity of denicotinized ("reduced exposure") cigarettes was equivalent to 3R4F (or worse).

Conclusions: This study provides a detailed analysis of Nrf2-related cytoprotective mechanisms activated in response to 3R4F and ULN-derived TS exposure correlating the results with their oxidative and inflammatory potential. Toxicants present in soluble cigarette smoke extracts (CSE) and not nicotine seem to be the primary determinant of vascular toxicity. In this respect our results from this and previous studies suggest that chronic TS exposure can overcome Nrf2 and NFκB-p65 dependent cytoprotective mechanisms of the brain microvascular endothelium possibly leading to BBB impairment and loss of BBB integrity.

No MeSH data available.


Related in: MedlinePlus