The role of Nrf2 dependent antioxidant response pathway is well established in disorders aggravated by cigarette smoke such as lung emphysema, COPD, atherosclerosis and cardiac dysfunction [23–25]. In these disorders Nrf2 deficiency has clearly been demonstrated to increase the susceptibility to cellular toxicity [26] and several natural and/or synthetic antioxidant based supplemental therapies have proven to be beneficial in ameliorating the oxidative damage [27, 28].

In this study we assessed the activity and biological responses of the human BBB endothelium to a number of biological factors related to Nrf2 activation. The endpoint goal of this work was to unravel (at a molecular level) the cause–effect relationship between the oxidative potential of full flavor (3R4F) and “reduced exposure” (ULN) tobacco products and the biological response of these cells.

As expected the biological response of the BBB endothelium in respect to Nrf2 expression/activity supported our original hypothesis of a direct correlation between the oxidative potential of a tobacco product and its cellular toxicity. This is clearly shown in Figure 1 demonstrating a striking relationship between oxidative stress generated by the CSE extracts on cultured endothelial cells (Figure 1a) and the effect on the expression level and nuclear translocation of Nrf2 in the same cells (Figure 1d). A data that already argue with the notion of “reduced exposure” tobacco product (such as ULN) being intrinsically less harmful than conventional cigarettes (such as 3R4F). Our results also indicate that cigarette reduction in nicotine content has a negligible or at best a very modest effect on the overall “safety” of the product itself in terms of oxidative damage to cells and tissues.

Nrf2-dependent anti-oxidant response is mainly divided into detoxification enzymes (such as Phase I—oxidation/reduction, Phase II—conjugation enzymes and Phase III—efflux transporters) and anti-oxidant based system (such as glutathione and thioredoxin) [11]. As a follow up study, we performed transcriptome analysis to screen for the expression of several downstream targets of Nrf2-dependent antioxidant response. Nrf2 dependent downstream genes were also highly up-regulated in response to CSE exposure whereas nicotine did not bear any significant effect. Specifically, a number of Phase I detoxification genes depending upon Nrf2/ARE pathway activation were up-regulated including NQO1 (see also Additional file 1: Table S1). NQO1 is a cytoplasmic 2-electron reductase that prevents the reduction of quinones and the resulting production of radical species. NQO1 response to CSE exposure was observed at the gene expression, transcription and protein expression levels (see Figure 2). Taken together, we show that the both conventional and “reduced exposure” tobacco products (but not nicotine) elicit a strong oxidative stress response in BBB endothelial cells that well correlate with Nrf2 activation also elicited by CSE exposure.

Expression of Phase I Cytochrome P450s genes such as CYP2S1 and CYP51A1 were also up-regulated by exposure to CSE from both cigarette products although a corresponding increase in protein expression was not evident (see Figure 3). This could be possibly due to a longer time requirement for the gene expression changes to translate into measurable alteration of protein expression. Important to note however, is that very few studies have reported strong baseline expression of CYPs in normal BBB endothelium [29]. Thus, a distinct pattern of gene up-regulation of both CYP2S1 and CYP51A1 which have been observed in response to TS detoxification is a relevant piece of information further supporting the involvement of Nrf2 -dependent cytoprotection in response to TS exposure (see also Additional file 1: Table S2).

Another important component of BBB is the class of ABC transporters that function to efflux out both endogenous and exogenous toxicants out of the brain. As shown in Figure 4a, our data provides novel evidence of a potential augmentation of two classical ABCs, ABCA1 (P-gp) and ABCC4 (MRP4) at the BBB in response to CSE exposure. These changes were tangible enough to show at the gene expression level however were too small to be detected to a significant level of confidence at the transcription and translation level. Nevertheless functional assessment of an assessment of functional efflux activity specifically for P-gp (measured via Rhodamine-123; see also Figure 4c) clearly demonstrated statistically significant higher efflux function in EC cultures exposed to 3R4F. ULN effect was more modest. Considering that nicotine mildly down-regulated P-gp gene expression and did not affect its efflux activity, the difference between 3R4F and ULN in respect to P-gp activity can possibly reflect a difference in cigarette composition and substances released in solution which may differently affect P-gp expression. This provided a reasonable explanation (although difficult to ascertain given the complexity of cigarette composition) where again the total oxidative capacity of the TS product was the determining factor of cellular toxicity and activation of cytoprotective mechanisms. In addition to these major drug efflux transporters, gene array screening revealed up-regulation of several other ABC transporters primarily involved in cholesterol trafficking across the BBB (See also Additional file 1: Table S3).

The next level of cytoprotection provided by Nrf2-ARE pathway is the heightened synthesis of anti-oxidants such as GSH, thioredoxin. Exposure to CSE from 3R4F and ULN both elicited gene transcription and increase in the protein expression levels of the Cystine/glutamate transporter antiporter SLC7A11 and the Glutamate cysteine ligase regulatory subunit (also known as gamma-glutamyl cysteine synthetase) which is the first rate limiting enzyme of glutathione (GSH) synthesis (Figure 5a–c). Note, in fact, how the GSH//GSSG rate reflects the level of oxidative stress caused by TS exposure leading to GSH depletion (most evident in BBB endothelial cultures exposed to 3R4F; see Figure 5d). Note also the nicotine is capable in some measure to elicit oxidative stress. This is also reflected in the more modest but still significant decrease of GSH/GSSG ratio observed in BBB endothelial cells exposed to nicotine. Although the effect is relatively modest, this can explain while 3R4F exposure determined the most significant impact in terms of GSH reduction when compared to ULN product. ULN contain similar amount of TAR but negligible amount of nicotine. In addition, concomitant up-regulation of several other genes related to NAPDH production were also observed. (See Additional file 1: Table S4). These genes function to recycle and regenerate the anti-oxidants back to the inactive state [11].

Oxidative and inflammatory changes following TS exposure have been reported to elevate stress inducible enzyme HMOX-1 [30, 31]. Both 3R4F and ULN products showed comparable alterations in HMOX-1 (see Figure 6) and related genes (such as pirin, ferritin). Interestingly, apart from metal chelation; pirin is also reported to govern the activation of NFκβ and its related genes via Nrf2 [21]. It is a non heme Fe protein that may function to sense redox stress for the NFκβ pro-inflammatory signalling and govern the expression of downstream genes involved in immune responses [21, 32, 33]. Ferritin instead is a major intracellular storage protein which helps with iron homeostasis. It is usually composed of various ratios of light chain and heavy chain subunits. Following CSE treatment we observed increased transcription of several of these subunits whereas, both 3R4F and ULN products led to the activation of genes related to iron sequestration. In contrast, nicotine failed to induce any changes in gene and/or protein expression of HMOX-1. This again reiterated that the level of BBB toxicity was dependent on the oxidative and/or inflammatory capacity of the TS product which may not be discriminated by the nicotine content alone.

Apart from Nrf2, another cell survival transcription factor reported in event of oxidative stress is nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB); a protein complex that controls DNA transcription and is involved in cellular responses to harmful stimuli (including free radicals, pro-inflammatory cytokines, ultraviolet irradiation, etc.) and plays a key role in regulating the immune and cell survival [22]. NFκB-p65 not only increased in total cytosolic levels, but also showed increased translocation into the nucleus (See Figure 7) in response to CSE exposure. Apart from classical activation of NFκβ in events of inflammatory/oxidative stress, Nrf1/Nrf2 can up-regulate GCLC transcription indirectly via modulation of levels of NFκβ [34, 35]. Although based on current results we cannot assertively state if NFκB-p65 was directly or indirectly activated; we observed a significant increase of its nuclear translocation following CSE exposure. The effect was particularly enhanced in response to ULN-derived CSE; a cigarette product considered “reduced exposure” and less harmful than conventional products. In addition, NFκβ-p65 expression and translocation patterns consistently paralleled stress fighting responses as reported in earlier observations. NFκβ activation typically involves either canonical or non-canonical pathways. The selection dependents on whether p50/p65 (RelA) (canonical) or p52/RelB (non-canonical) is involved in the translocation and downstream activation. We could clearly observe increased translocation of the p65 sub unit into the nucleus indicating activation of the canonical pathway and associated pro-inflammatory/cell survival responses. At this point, it is difficult assert if only this pathway was associated with the TS toxicity coping mechanisms; further studies will be required in future to specifically address this conundrum. Furthermore, we observed an increase of IL-8 release following both 3R4F and ULN-derived CSE exposures. In agreement with our findings, p65 sub unit was reported to activate IL-8 expression via promoter binding [36].

Interestingly, previous in vitro studies by Barr et al. [37] have shown a dose dependent nicotine-induced activation of stress-dependent NFkB pathway in mesencephalic cells. However, we observed only a marginal response to nicotine exposure (including NFκβ-p65 protein expression and nuclear translocation). A possible explanation of this discrepancy could be attributed to two factors: (1) for our experiments the reference concentration of nicotine used was approximately 0.6 µM which falls to the lower end of the concentrations tested by Barr and co-workers; (2) time of assessment. In our study, oxidative stress measurements (as shown in Figure 1) were measured at 3 h at which point we did note a slight increase of oxidative stress in nicotine-exposed cultures versus controls. However, measurements of downstream effects such as NFkB-p65 activation (expression and nuclear translocation) were assessed at 24 h post exposure. It is possible that the oxidative stress effects elicited by nicotine fade off and became negligible at that time point.

As a part of vascular inflammation; cigarette-toxicity can lead to the up-regulation of several chemokines that promote cellular adhesion of white blood cells and facilitate their extravasation across the brain vasculature [38]. To understand the extent of vascular inflammation produced in the ECs following CSE exposure; we measured two major chemokines (IL-8 and MCP-1). Both these chemokines have been reported to be involved in the chemotaxis of neutrophils and monocytes after cigarette smoke exposure in lung and other peripheral vasculatures [39–41]. Release of IL-8 and MCP-1 was significantly increased in BBB endothelial cultures exposed to CSE (both 3R4F and ULN). This is in agreement with earlier published results by our group demonstrating a number of inflammatory changes (including IL-6 release and up-regulation of vascular adhesion molecules such as VCAM-1, Pecam-1) in BBB ECs exposed to TS [8]. Finally, CSE from both 3R4F and ULN elicited endothelial release of VEGF, a known modulator of vascular angiogenesis released in response to hypoxic condition and implicated in the alteration of BBB vascular integrity [42, 43].