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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 ABC efflux transporter expression and functionality in hCMEC/D3 cell line. Cells were exposed to nicotine (100 ng/mL) or CSE derived from 3R4F or ULN cigarettes. a P-gp efflux activity was determined by intracellular accumulation of rhodamine123 (a P-gp substrate) efflux, as an indirect correlate of P-gp activity n = 3/condition and replicated twice. b Transcriptome analysis of ABC efflux transporters, ABCB1 (P-gp) and ABCC4 (MRP4) following treatment (n = 6 biological replicates); c RT-PCR analysis of mRNA expression of P-gp and ABCC4 in hCMEC/D3 cells (n = 6 biological replicates); western blot analysis of transporter protein expression (n = 6 biological replicates). 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, vs. control.
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Fig4: Effects of CSE on ABC efflux transporter expression and functionality in hCMEC/D3 cell line. Cells were exposed to nicotine (100 ng/mL) or CSE derived from 3R4F or ULN cigarettes. a P-gp efflux activity was determined by intracellular accumulation of rhodamine123 (a P-gp substrate) efflux, as an indirect correlate of P-gp activity n = 3/condition and replicated twice. b Transcriptome analysis of ABC efflux transporters, ABCB1 (P-gp) and ABCC4 (MRP4) following treatment (n = 6 biological replicates); c RT-PCR analysis of mRNA expression of P-gp and ABCC4 in hCMEC/D3 cells (n = 6 biological replicates); western blot analysis of transporter protein expression (n = 6 biological replicates). 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, vs. control.

Mentions: Blood brain barrier endothelial cells are highly enriched with polarized expression of ATP-binding cassette superfamily of drug efflux transporters such as P-glycoprotein (P-gp) that prevent the brain penetration and accumulation of toxic substances including xenobiotics. Previous studies have shown that Nrf2 activation induces the transcription of major drug efflux transporters [11]. As shown in Figure 4, microarray analysis revealed a significant up-regulation of two major ABC efflux transporters, P-glycoprotein (P-gp; ABCB1) and multidrug resistant protein-4 (MRP-4; ABCC4; p < 0.05, vs. control) in response to 3R4F exposure. The biological effect is consistent with a marked increase in rhodamine123 efflux (p < 0.05, vs. control) used as a measure of P-gp functional activity. CSE from ULN also increased the gene expression of P-gp (p < 0.01 vs. control), but not that of MRP4 (Figure 4b). However, RT-PCR and western blotting analysis of the cellular membrane fractions did not show a corresponding increase in gene transcription and/or protein expression levels for either of these transporters (Figure 4c). Interestingly, when compared to nicotine, TS did produce a statistically significant increase of P-gp transcription when compared to nicotine since surprisingly nicotine caused a modest reduction in P-gp transcription when compared to controls.Figure 4


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 ABC efflux transporter expression and functionality in hCMEC/D3 cell line. Cells were exposed to nicotine (100 ng/mL) or CSE derived from 3R4F or ULN cigarettes. a P-gp efflux activity was determined by intracellular accumulation of rhodamine123 (a P-gp substrate) efflux, as an indirect correlate of P-gp activity n = 3/condition and replicated twice. b Transcriptome analysis of ABC efflux transporters, ABCB1 (P-gp) and ABCC4 (MRP4) following treatment (n = 6 biological replicates); c RT-PCR analysis of mRNA expression of P-gp and ABCC4 in hCMEC/D3 cells (n = 6 biological replicates); western blot analysis of transporter protein expression (n = 6 biological replicates). 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, vs. control.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4477310&req=5

Fig4: Effects of CSE on ABC efflux transporter expression and functionality in hCMEC/D3 cell line. Cells were exposed to nicotine (100 ng/mL) or CSE derived from 3R4F or ULN cigarettes. a P-gp efflux activity was determined by intracellular accumulation of rhodamine123 (a P-gp substrate) efflux, as an indirect correlate of P-gp activity n = 3/condition and replicated twice. b Transcriptome analysis of ABC efflux transporters, ABCB1 (P-gp) and ABCC4 (MRP4) following treatment (n = 6 biological replicates); c RT-PCR analysis of mRNA expression of P-gp and ABCC4 in hCMEC/D3 cells (n = 6 biological replicates); western blot analysis of transporter protein expression (n = 6 biological replicates). 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, vs. control.
Mentions: Blood brain barrier endothelial cells are highly enriched with polarized expression of ATP-binding cassette superfamily of drug efflux transporters such as P-glycoprotein (P-gp) that prevent the brain penetration and accumulation of toxic substances including xenobiotics. Previous studies have shown that Nrf2 activation induces the transcription of major drug efflux transporters [11]. As shown in Figure 4, microarray analysis revealed a significant up-regulation of two major ABC efflux transporters, P-glycoprotein (P-gp; ABCB1) and multidrug resistant protein-4 (MRP-4; ABCC4; p < 0.05, vs. control) in response to 3R4F exposure. The biological effect is consistent with a marked increase in rhodamine123 efflux (p < 0.05, vs. control) used as a measure of P-gp functional activity. CSE from ULN also increased the gene expression of P-gp (p < 0.01 vs. control), but not that of MRP4 (Figure 4b). However, RT-PCR and western blotting analysis of the cellular membrane fractions did not show a corresponding increase in gene transcription and/or protein expression levels for either of these transporters (Figure 4c). Interestingly, when compared to nicotine, TS did produce a statistically significant increase of P-gp transcription when compared to nicotine since surprisingly nicotine caused a modest reduction in P-gp transcription when compared to controls.Figure 4

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