Effects of unburned tobacco smoke on inflammatory and oxidative mediators in the rat prefrontal cortex

Abstract

Although the Food and Drug Administration has authorized the marketing of "heat-not-burn" (HnB) electronic cigarettes as a modified risk tobacco product (MRTP), toxicological effects of HnB smoke exposure on the brain are still unexplored. Here, paramagnetic resonance of the prefrontal cortex (PFC) of HnB-exposed rats shows a dramatic increase in reactive radical species (RRS) yield coupled with an inflammatory response mediated by NF-κB-target genes including TNF-α, IL-1β, and IL-6 and the downregulation of peroxisome proliferator-activated receptor (PPAR) alpha and gamma expression. The PFC shows higher levels of 8-hydroxyguanosine, a marker of DNA oxidative damage, along with the activation of antioxidant machinery and DNA repair systems, including xeroderma pigmentosum group C (XPC) protein complex and 8-oxoguanine DNA glycosylase 1. HnB also induces the expression of drug-metabolizing enzymes such as CYP1A1, CYP2A6, CYP2B6, and CYP2E, particularly involved in the biotransformation of nicotine and several carcinogenic agents such as aldehydes and polycyclic aromatic hydrocarbons here recorded in the HnB stick smoke. Taken together, these effects, from disruption of redox homeostasis, inflammation, PPAR manipulation along with enhanced bioactivation of neurotoxicants, and upregulation of cMYC protooncogene to impairment of primary cellular defense mechanisms, suggest a possible increased risk of brain cancer. Although the HnB device reduces the emission of tobacco toxicants, our findings indicate that its consumption may carry a risk of potential adverse health effects, especially in non-smokers so far. Further studies are needed to fully understand the long-term effects of these devices.

Keywords: KDMs; e-cigarette; heat-not-burn; inflammation; oxidative stress; peroxisome proliferator-activated receptors; prefrontal cortex.

FIGURE 1

Chemical analysis of HnB smoke. Data show the presence of toxic aldehydes (A), polycyclic aromatic hydrocarbons (B), and volatile organic compounds (C). Data are expressed as the means ± SD of at least two replicates from two independent experiments.

FIGURE 2

HnB exposure leads to oxidative stress and induces the NRF2-mediated antioxidant response. Radical species content, here measured through the intensity of the first spectral line of electronic paramagnetic resonance (EPR) (arbitrary units), is significantly higher in samples from exposed animals compared to those from controls (A). HnB group showed a significant increase in NRF2 (∼65 kDa) (B), with an upregulation of CAT (∼55 kDa) (C) and SOD-1, here shown as a relative gene expression (D) and protein expression (∼19 kDa) (E). A representative blot is reported under the histograms. Bars represent the means ± SEM; ^* p < 0.05, ^** p < 0.01; two-tailed t-test.

FIGURE 3

Animals exposed to HnB mainstream smoke show higher levels of 8-OHdG, a DNA oxidative damage marker, along with the activation of the DNA repair machinery through the induction of XPC and OGG-1 proteins. Exposed animals show higher levels of 8-OHdG, an oxidative damage marker, which is measured through ELISA assay and expressed as pg of 8-dG/mLOH (A). DNA repair systems are induced in the exposed group: XPC (∼26 kDa) (B) and OGG-1 (∼47 kDa) (C) play a key role in nucleotide excision repair and base excision repair. They are significantly upregulated in the PFC of the exposed rats compared to controls. No significant changes in the H2AX (∼17 kDa) phosphorylation rate were recorded (D). A representative blot is reported under the histograms. Bars represent the means ± SEM; ^* p < 0.05, ^** p < 0.01; two-tailed t-test.

FIGURE 4

HnB exposure induces the expression of CYPs. The exposure to HnB smoke led to an overexpression of CYP1A1 (∼63 kDa) activating aromatic amines, dioxins, and PAHs (A). CYP2A6 (∼56 kDa) (involved in metabolic activation of carcinogenic nitrosamines in tobacco smoke as well as nicotine metabolism); (B); CYP2B6 (∼66 kDa) (activating bupropion smoking-cessation drug); (C) and CYP2E1 (∼50 kDa) (activating alcohol, nitrosamines, benzene, acetone, and acrylamide) (D). A representative blot is reported under the histograms. Bars represent the means ± SEM; ^* p < 0.05, ^** p < 0.01; two-tailed t-test.

FIGURE 5

HnB exposure induces the expression of protooncogene c-MYC. Animals exposed to HnB smoke showed an increase in protooncogene c-MYC (∼49 kDa). A representative blot is reported under the histograms. Bars represent the means ± SEM; ^**** p < 0.0001; two-tailed t-test.

FIGURE 6

HnB activates NF-κB-mediated inflammatory response. Exposed animals reported an increase in phosphorylation of NF-κB (p65 Ser 536) (∼61 kDa) (A), along with the upregulation of TNF-α (∼25 kDa) (B), IL-1β (∼17 kDa) (C), and IL-6 (∼26 kDa) (D). On the contrary, no significant changes in IL-8 (∼11 kDa) (E) expression were observed. A representative blot is reported under the histograms. Bars represent the means ± SEM; ^* p < 0.05; two-tailed t-test.

FIGURE 7

Effects of the HnB mainstream on the relative gene expression of KDM6A (A), PPARα (B), and PPARγ (C) in the rat PFC. Data represent 2^−ΔΔCT values calculated using the ΔΔCT method and are expressed as the means ± SEM; ^* p < 0.05; ^** p < 0.01, two-tailed t-test.

FIGURE 8

Schematic representation of the effects of HnB smoking on oxidative stress, inflammatory process, and DNA damage. A large number of compounds found in the HnB mainstream are oxidants or chemicals that can be bioactivated by CYP to mutagens and ultimate carcinogens (via carcinogenesis) (center). Many of them are known inducers of CYP (e.g., aldehydes and PAHs), a phenomenon typically associated with both a greater bioactivation of ubiquitous pre-mutagens/pre-carcinogens and an increase in the generation of ROS (via co-carcinogenesis) (left side). Both processes can increase the risk of cancer, the former directly due to the effect of smoke compounds and the latter indirectly through the upregulation determined by these same compounds. Moreover, we can assume that oxidative stress promoted neuroinflammatory processes through an upregulation of histone demethylase KDM6A by increasing the target NF-κB pro-inflammatory mediators, such as IL-6 and IL-1β, together with the downregulation of PPAR expression as negative regulators of oxidative stress-induced inflammation (right side).