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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 potentiates oxidative stress responses in BBB endothelial cells. Briefly, confluent hCMEC/D3 cell monolayers were exposed to media containing nicotine only (100 μg/mL) or CSEs from full flavor (3R4F) or ULN tobacco products containing nicotine equivalent to 100 ng/mL). Fresh media without nicotine or mainstream TS served as controls. a Immunofluorescence analysis of cellular ROS levels (an indicator of oxidative stress load) were determined by using CellROX® Green reagent following exposure to CSE or nicotine treatment (3 h); b gene array based analysis of Nrf2 gene expression changes was determined after control, nicotine or TS exposure (n = 6); c Nrf2 mRNA expression was quantified by RT-PCR using primer specific sequences (n = 4); d Nrf2 expression and nuclear translocation following exposure to different conditions at early time point of 8 h as analyzed by western blotting. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control; n = 4 biological replicates). *p < 0.05 vs. control.
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Fig1: CSE exposure potentiates oxidative stress responses in BBB endothelial cells. Briefly, confluent hCMEC/D3 cell monolayers were exposed to media containing nicotine only (100 μg/mL) or CSEs from full flavor (3R4F) or ULN tobacco products containing nicotine equivalent to 100 ng/mL). Fresh media without nicotine or mainstream TS served as controls. a Immunofluorescence analysis of cellular ROS levels (an indicator of oxidative stress load) were determined by using CellROX® Green reagent following exposure to CSE or nicotine treatment (3 h); b gene array based analysis of Nrf2 gene expression changes was determined after control, nicotine or TS exposure (n = 6); c Nrf2 mRNA expression was quantified by RT-PCR using primer specific sequences (n = 4); d Nrf2 expression and nuclear translocation following exposure to different conditions at early time point of 8 h as analyzed by western blotting. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control; n = 4 biological replicates). *p < 0.05 vs. control.

Mentions: Main and side stream TS contain high levels of toxic reactive aldehydes and pro-oxidants that are shown to destabilize the cellular redox balance. We assessed the impact of TS generated from different tobacco products, on cellular ROS levels (oxidative stress load) in BBB endothelium using CellROX® Green Reagent. As shown in Figure 1a, exposure to CSE from both full flavor (3R4F) and ULN cigarettes rapidly increased endothelial ROS (as early as 3 h post-exposure), with greater intensity of fluorescence, compared to control. In comparison, exposure to nicotine elicited only a mild oxidative response (Figure 1a).Figure 1


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 potentiates oxidative stress responses in BBB endothelial cells. Briefly, confluent hCMEC/D3 cell monolayers were exposed to media containing nicotine only (100 μg/mL) or CSEs from full flavor (3R4F) or ULN tobacco products containing nicotine equivalent to 100 ng/mL). Fresh media without nicotine or mainstream TS served as controls. a Immunofluorescence analysis of cellular ROS levels (an indicator of oxidative stress load) were determined by using CellROX® Green reagent following exposure to CSE or nicotine treatment (3 h); b gene array based analysis of Nrf2 gene expression changes was determined after control, nicotine or TS exposure (n = 6); c Nrf2 mRNA expression was quantified by RT-PCR using primer specific sequences (n = 4); d Nrf2 expression and nuclear translocation following exposure to different conditions at early time point of 8 h as analyzed by western blotting. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control; n = 4 biological replicates). *p < 0.05 vs. control.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: CSE exposure potentiates oxidative stress responses in BBB endothelial cells. Briefly, confluent hCMEC/D3 cell monolayers were exposed to media containing nicotine only (100 μg/mL) or CSEs from full flavor (3R4F) or ULN tobacco products containing nicotine equivalent to 100 ng/mL). Fresh media without nicotine or mainstream TS served as controls. a Immunofluorescence analysis of cellular ROS levels (an indicator of oxidative stress load) were determined by using CellROX® Green reagent following exposure to CSE or nicotine treatment (3 h); b gene array based analysis of Nrf2 gene expression changes was determined after control, nicotine or TS exposure (n = 6); c Nrf2 mRNA expression was quantified by RT-PCR using primer specific sequences (n = 4); d Nrf2 expression and nuclear translocation following exposure to different conditions at early time point of 8 h as analyzed by western blotting. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control; n = 4 biological replicates). *p < 0.05 vs. control.
Mentions: Main and side stream TS contain high levels of toxic reactive aldehydes and pro-oxidants that are shown to destabilize the cellular redox balance. We assessed the impact of TS generated from different tobacco products, on cellular ROS levels (oxidative stress load) in BBB endothelium using CellROX® Green Reagent. As shown in Figure 1a, exposure to CSE from both full flavor (3R4F) and ULN cigarettes rapidly increased endothelial ROS (as early as 3 h post-exposure), with greater intensity of fluorescence, compared to control. In comparison, exposure to nicotine elicited only a mild oxidative response (Figure 1a).Figure 1

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