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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 GSH based antioxidant system. a Microarray studies revealed an up-regulation in gene expression of two major genes involved in GSH synthesis, SLC7A11 and GCLM; b quantitative assessment using real-time PCR and western blot analyses confirm microarray analysis. c mRNA expression of modifier and catalytic subunits of GLC and protein expression of GCLC and GCLM following CSE exposure derived from 3R4F and ULN products including nicotine. Representative western blots were shown with actin as a loading control; d GSH/GSSG ratio was determined using Thiol green reagent, as mentioned in the methods section. 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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Fig5: Effects of CSE on GSH based antioxidant system. a Microarray studies revealed an up-regulation in gene expression of two major genes involved in GSH synthesis, SLC7A11 and GCLM; b quantitative assessment using real-time PCR and western blot analyses confirm microarray analysis. c mRNA expression of modifier and catalytic subunits of GLC and protein expression of GCLC and GCLM following CSE exposure derived from 3R4F and ULN products including nicotine. Representative western blots were shown with actin as a loading control; d GSH/GSSG ratio was determined using Thiol green reagent, as mentioned in the methods section. 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: The next major component of the Nrf2-ARE pathway is the anti-oxidant system. In this study, we compared the effects of CSE derived from conventional and reduced exposure products on the genes involved in synthesis of major anti-oxidant glutathione (GSH), such as glutathione cysteine ligase and SLC7A11 [20]. Genome wide transcriptional profiling revealed an amplified gene expression of GCL-modifier unit (GCLM) and SLC7A11 in BBB endothelial cells exposed to CSE from 3R4F and ULN (Figure 5a). These results were further validated by RT-PCR analysis indicating a potential up-regulation of SLC7A11 (Figure 5b, p < 0.05 and p < 0.01 vs. control) and GCLM transcription (Figure 5c, p < 0.05 vs. control) by 3R4F and ULN smoke extracts, respectively. Moreover, alterations in the protein expression of SLC7A11 and GCLM followed a similar trend with significant increase upon exposure to CSE derived from both products (Figure 5b, c). In addition, 3R4F but not ULN moderately increased gene transcription of GCL-catalytic subunit (GCLC), while GCLC protein expression was up-regulated by both products to a similar extent (Figure 5c). By contrast, nicotine did not produced any significant alterations in GCLM and SLC7A11 gene expression, although we observed a marked increase in the cytoplasmic expression of the catalytic and modifier subunits of GCL following nicotine exposure, as shown by western blotting (Figure 5c). Importantly, all the tested products (including nicotine) reduced the turn-over rate of cellular GSH, as demonstrated by the GSH/GSSG ratio (Figure 5d, p < 0.05). This clearly suggests a depletion of cellular antioxidant protection against incumbent oxidative stress load caused by the exposure to smoke extracts and nicotine (in smaller measure).Figure 5


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 GSH based antioxidant system. a Microarray studies revealed an up-regulation in gene expression of two major genes involved in GSH synthesis, SLC7A11 and GCLM; b quantitative assessment using real-time PCR and western blot analyses confirm microarray analysis. c mRNA expression of modifier and catalytic subunits of GLC and protein expression of GCLC and GCLM following CSE exposure derived from 3R4F and ULN products including nicotine. Representative western blots were shown with actin as a loading control; d GSH/GSSG ratio was determined using Thiol green reagent, as mentioned in the methods section. 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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Fig5: Effects of CSE on GSH based antioxidant system. a Microarray studies revealed an up-regulation in gene expression of two major genes involved in GSH synthesis, SLC7A11 and GCLM; b quantitative assessment using real-time PCR and western blot analyses confirm microarray analysis. c mRNA expression of modifier and catalytic subunits of GLC and protein expression of GCLC and GCLM following CSE exposure derived from 3R4F and ULN products including nicotine. Representative western blots were shown with actin as a loading control; d GSH/GSSG ratio was determined using Thiol green reagent, as mentioned in the methods section. 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: The next major component of the Nrf2-ARE pathway is the anti-oxidant system. In this study, we compared the effects of CSE derived from conventional and reduced exposure products on the genes involved in synthesis of major anti-oxidant glutathione (GSH), such as glutathione cysteine ligase and SLC7A11 [20]. Genome wide transcriptional profiling revealed an amplified gene expression of GCL-modifier unit (GCLM) and SLC7A11 in BBB endothelial cells exposed to CSE from 3R4F and ULN (Figure 5a). These results were further validated by RT-PCR analysis indicating a potential up-regulation of SLC7A11 (Figure 5b, p < 0.05 and p < 0.01 vs. control) and GCLM transcription (Figure 5c, p < 0.05 vs. control) by 3R4F and ULN smoke extracts, respectively. Moreover, alterations in the protein expression of SLC7A11 and GCLM followed a similar trend with significant increase upon exposure to CSE derived from both products (Figure 5b, c). In addition, 3R4F but not ULN moderately increased gene transcription of GCL-catalytic subunit (GCLC), while GCLC protein expression was up-regulated by both products to a similar extent (Figure 5c). By contrast, nicotine did not produced any significant alterations in GCLM and SLC7A11 gene expression, although we observed a marked increase in the cytoplasmic expression of the catalytic and modifier subunits of GCL following nicotine exposure, as shown by western blotting (Figure 5c). Importantly, all the tested products (including nicotine) reduced the turn-over rate of cellular GSH, as demonstrated by the GSH/GSSG ratio (Figure 5d, p < 0.05). This clearly suggests a depletion of cellular antioxidant protection against incumbent oxidative stress load caused by the exposure to smoke extracts and nicotine (in smaller measure).Figure 5

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