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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 exposure (24 h) on BBB endothelial gene and protein expression of HMOX1. a Several genes belonging to HMOX-1 and heme recycling were up-regulated following CSE exposure of TS products. Nicotine did not produce any change in these genes; b real time RT-PCR showed up-regulation of HMOX-1 protein expression following exposure to full flavor, and ULN CSEs; c western blot analyses of cellular membrane fractions corroborate with mRNA changes showing statistically significant up-regulation in protein expression; representative western blots were shown with actin as a loading control; d immunofluorescence analysis of HMOX1 expression. 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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Fig6: Effects of CSE exposure (24 h) on BBB endothelial gene and protein expression of HMOX1. a Several genes belonging to HMOX-1 and heme recycling were up-regulated following CSE exposure of TS products. Nicotine did not produce any change in these genes; b real time RT-PCR showed up-regulation of HMOX-1 protein expression following exposure to full flavor, and ULN CSEs; c western blot analyses of cellular membrane fractions corroborate with mRNA changes showing statistically significant up-regulation in protein expression; representative western blots were shown with actin as a loading control; d immunofluorescence analysis of HMOX1 expression. 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: Continuing on the same path, we assessed the effects of nicotine, 3R4F and ULN exposure on gene expression, transcription and translation of heme oxygenase (HMOX-1). HMOX-1 is also a component of the cellular antioxidant cytoprotective mechanisms along with other molecular networks (including ferritin and pirin involved in iron/metal sequestration) [21]. As shown in Figure 6a, transcriptome analysis revealed a significant increase in the gene expression levels of HMOX-1 and other anti-oxidant molecules in response to 3R4F and ULN exposure. Results were corroborated by RT-PCR and western blot analyses in line with gene expression data as shown in Figure 6b, c respectively (p < 0.05, vs. control). Immunofluorescence analysis of the BBB endothelial monolayers further supports these observations (Figure 6d).Figure 6


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 exposure (24 h) on BBB endothelial gene and protein expression of HMOX1. a Several genes belonging to HMOX-1 and heme recycling were up-regulated following CSE exposure of TS products. Nicotine did not produce any change in these genes; b real time RT-PCR showed up-regulation of HMOX-1 protein expression following exposure to full flavor, and ULN CSEs; c western blot analyses of cellular membrane fractions corroborate with mRNA changes showing statistically significant up-regulation in protein expression; representative western blots were shown with actin as a loading control; d immunofluorescence analysis of HMOX1 expression. 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
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getmorefigures.php?uid=PMC4477310&req=5

Fig6: Effects of CSE exposure (24 h) on BBB endothelial gene and protein expression of HMOX1. a Several genes belonging to HMOX-1 and heme recycling were up-regulated following CSE exposure of TS products. Nicotine did not produce any change in these genes; b real time RT-PCR showed up-regulation of HMOX-1 protein expression following exposure to full flavor, and ULN CSEs; c western blot analyses of cellular membrane fractions corroborate with mRNA changes showing statistically significant up-regulation in protein expression; representative western blots were shown with actin as a loading control; d immunofluorescence analysis of HMOX1 expression. 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: Continuing on the same path, we assessed the effects of nicotine, 3R4F and ULN exposure on gene expression, transcription and translation of heme oxygenase (HMOX-1). HMOX-1 is also a component of the cellular antioxidant cytoprotective mechanisms along with other molecular networks (including ferritin and pirin involved in iron/metal sequestration) [21]. As shown in Figure 6a, transcriptome analysis revealed a significant increase in the gene expression levels of HMOX-1 and other anti-oxidant molecules in response to 3R4F and ULN exposure. Results were corroborated by RT-PCR and western blot analyses in line with gene expression data as shown in Figure 6b, c respectively (p < 0.05, vs. control). Immunofluorescence analysis of the BBB endothelial monolayers further supports these observations (Figure 6d).Figure 6

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