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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

Effects of CSE on Phase I detoxification genes. HCMEC/D3 cells were exposed to nicotine or CSE from different products (see “Methods”). Effects of CSE and nicotine on NQO-1 mRNA and protein expression were characterized by: a1 gene array analysis; a2 real-time RT-PCR; b immunofluorescence and western blot analyses of NQO-1 protein. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ****p < 0.0001 vs. control. n = 6/biological replicates/condition.
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Fig2: Effects of CSE on Phase I detoxification genes. HCMEC/D3 cells were exposed to nicotine or CSE from different products (see “Methods”). Effects of CSE and nicotine on NQO-1 mRNA and protein expression were characterized by: a1 gene array analysis; a2 real-time RT-PCR; b immunofluorescence and western blot analyses of NQO-1 protein. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ****p < 0.0001 vs. control. n = 6/biological replicates/condition.

Mentions: As shown in Figure 2a, both microarray and RT-PCR analyses revealed a significant induction of NQO1 gene (a major enzyme of Phase I detoxification) both by 3R4F and ULN products (p < 0.01 and p < 0.0001 vs control fold change respectively), but not nicotine. Accordingly, exposure to 3R4F and ULN upregulated NQO1 protein expression as demonstrated by immunofluorescence and western blotting (Figure 2b; p < 0.05 and p < 0.01 for 3R4F and ULN, respectively). Notably, the up-regulation of NQO1 expression by ULN was comparable to that achieved by 3R4F.Figure 2


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)

Effects of CSE on Phase I detoxification genes. HCMEC/D3 cells were exposed to nicotine or CSE from different products (see “Methods”). Effects of CSE and nicotine on NQO-1 mRNA and protein expression were characterized by: a1 gene array analysis; a2 real-time RT-PCR; b immunofluorescence and western blot analyses of NQO-1 protein. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ****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

Fig2: Effects of CSE on Phase I detoxification genes. HCMEC/D3 cells were exposed to nicotine or CSE from different products (see “Methods”). Effects of CSE and nicotine on NQO-1 mRNA and protein expression were characterized by: a1 gene array analysis; a2 real-time RT-PCR; b immunofluorescence and western blot analyses of NQO-1 protein. Representative western blots were shown with actin as a loading control. Data were expressed as mean ± SEM (fold change over control). *p < 0.05, **p < 0.01, ****p < 0.0001 vs. control. n = 6/biological replicates/condition.
Mentions: As shown in Figure 2a, both microarray and RT-PCR analyses revealed a significant induction of NQO1 gene (a major enzyme of Phase I detoxification) both by 3R4F and ULN products (p < 0.01 and p < 0.0001 vs control fold change respectively), but not nicotine. Accordingly, exposure to 3R4F and ULN upregulated NQO1 protein expression as demonstrated by immunofluorescence and western blotting (Figure 2b; p < 0.05 and p < 0.01 for 3R4F and ULN, respectively). Notably, the up-regulation of NQO1 expression by ULN was comparable to that achieved by 3R4F.Figure 2

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