Cigarette smoke extract alters genome‐wide profiles of circular RNAs and mRNAs in primary human small airway epithelial cells

As a novel kind of non‐coding RNA, circular RNAs (circRNAs) were involved in various biological processes. However, the role of circRNAs in the developmental process of chronic obstructive pulmonary disease (COPD) is still unclear. In the present study, by using a cell model of COPD in primary human small airway epithelial cells (HSAECs) treated with or without cigarette smoke extract (CSE), we uncovered 4,379 previously unknown circRNAs in human cells and 903 smoke‐specific circRNAs, with the help of RNA‐sequencing and bioinformatic analysis. Moreover, 3,872 up‐ and 4,425 down‐regulated mRNAs were also identified under CSE stimulation. Furthermore, a putative circRNA‐microRNA‐mRNA network was constructed for in‐depth mechanism exploration, which indicated that differentially expressed circRNAs could influence expression of some key genes that participate in response to pentose phosphate pathway, ATP‐binding cassette (ABC) transporters, glycosaminoglycan biosynthesis pathway and cancer‐related pathways. Our research indicated that cigarette smoke had an influence on the biogenesis of circRNAs and mRNAs. CircRNAs might be involved in the response to CSE in COPD through the circRNA‐mediated ceRNA networks.     

Evaluation of urinary trans-3′-hydroxycotinine as a biomarker of children's environmental tobacco smoke exposure

Abstract

The utility of urinary trans-3′-hydroxy cotinine (3HC) as a biomarker of environmental tobacco smoke (ETS) exposure was investigated in comparison with urinary cotinine (COT), the sum (3HC?+?COT), and ratio of the two nicotine metabolites (3HC/COT). Participants were 150 ETS exposed children (aged 1–44 months) and their parents. Child urine samples were collected during 3weekly baseline assessments and at interviews administered 3, 6, 12, and 18 months after baseline. Findings indicate that 3HC and COT can be measured reliably (rho?=?0.96, 0.88) and show equivalent levels of repeated measures stability (rho?=?0.71, 0.75). COT, 3HC, and 3HC?+?COT showed equally strong associations with air nicotine levels, reported ETS contamination, and reported ETS exposure (r=0.60–0.70). The intraclass correlations of 3HC/COT were lower than those for COT or 3HC. Older children had a higher 3HC/COT ratio than younger children (3.5 versus 2.2), and non-Hispanic White children had a higher ratio than African-American children (3.2 versus 1.9). These findings suggest that COT, 3HC, and 3HC?+?COT are approximately equivalent and equally strong biomarkers of ETS exposure in children. Moreover, 3HC/COT may provide a useful indicator to investigate age- and race-related differences in the metabolism of COT and 3HC.     

Endogenous nitric oxide activation protects against cigarette smoking induced apoptosis in endothelial cells

Cigarette-induced endothelial dysfunction could be an early mediator of atherosclerosis. In this study, we explored the mechanisms of cigarette smoke extract (CSE)-induced human aortic endothelial cells (HAEC) apoptosis. We found that 10-65% of HAECs underwent apoptotic changes when HAECs were exposed to 0.001-0.02 cigarette equivalent unit of CSE for 4 h. CSE activated the caspases-3 and 8, the p38 MAP kinase and stress activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK). Specific inhibitors of p38 MAP or SAPK/JNK reduced CSE-induced caspase activation. We further showed that eNOS pre-activation by L-arginine reduced endothelial apoptosis from 65% to 5%; and eNOS inhibition by N-omega-nitro-L-arginine methyl ester accentuated CSE-induced endothelial apoptosis. We suggest that appropriate endogenous NO production may be an important protective mechanism against smoking-induced endothelial damage.     

Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 μM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a ⁵¹Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.     

The effect of concentrated smoke products on the restoration of highly disturbed mineral sands in southeast Victoria

Summary Recent studies have recognized the potential of broad‐scale surface application of smoke compounds for enhancing germination from the soil seed‐bank in fire‐prone vegetation communities. Results suggest that smoke technology may play, in the future, a significant role in the restoration and management of areas supporting indigenous vegetation. An important step in the development of smoke‐based restoration tools is the conduct of in situ field trials in a range of geographical locations and environmental conditions. However, most of the published work on the effectiveness of smoke products in promoting seedbank germination has been conducted at sites in southwestern Australia. The present study examines the effect of commercially available smoke‐water products on the regeneration of a highly disturbed former mine‐site at the Royal Botanic Gardens Cranbourne, in southeastern Victoria. Various combinations of concentrated smoke products and topsoil harvested from a nearby heathy woodland community were applied to exposed, uniform mineral sands to test their effect on seedling density and species richness of regrowth. The trials showed that after 12 months a number of common, herbaceous species including Austrodanthonia setacea, Opercularia varia and Platysace heterophylla were recorded in significantly higher numbers in areas treated with a commercial smoke‐water. However, there was no overall improvement in the density of seedlings or the richness of species as a result of the application of the smoke products. Similarly, total seedling density and species richness were not affected by the addition of topsoil, either alone or in combination with smoke products.     

Long-lasting occupancy of central nicotinic acetylcholine receptors after smoking: a PET study in monkeys

The aim of this study was to compare the degree of occupancy of central nicotinic acetylcholine receptors (nAChR) in isoflurane anaesthetized baboon brain following inhalation of tobacco smoke (one cigarette containing 0.9 mg nicotine) or i.v. nicotine (0.6 mg i.v.). [18F]Fluoro-A-85380 and positron emission tomography (PET) were used to assess the distribution volumes (DV) of the radiotracer in selected brain areas using a one-compartment model. Eighty minutes after nicotine i.v., DV was reduced by 50 and 66% in the thalamus and putamen, respectively. Six hours after nicotine, a reduction in DV (27% in the thalamus) was still observed. Eighty minutes after inhalation of tobacco smoke, DV was decreased by 52 and 65% in the thalamus and putamen, respectively. Previous PET experiments have demonstrated a short-lasting interaction of [11C]nicotine with nAChRs. Thus, we hypothesized that a metabolite of nicotine with high affinity and long half-live (several hours) could bind at nAChRs. Eighty minutes after a high dose of nornicotine (0.5 mg i.v.), DV was reduced by 53 and 31% in thalamus and putamen, respectively. No significant effect was observed following 0.15 mg nornicotine. Therefore, nornicotine could contribute to the long-lasting occupancy of central nAChRs after smoking.     

Gas Phase Oxidants of Cigarette Smoke Increase Nitric Oxide Synthase and Xanthine Oxidase Activities of Rabbit Brain Synaptosomes

In the present study we demonstrated that NO synthase and xanthine oxidase of synaptosomes isolated from rabbit brain cortex can be activated by the gas phase of cigarette smoke to produce nitric oxide and superoxide which react together to form peroxynitrite. Expose of synaptosomes, up to 3 hours, in the gas phase of cigarette smoke, a gradual increase in both nitric oxide and superoxide release that were inhibited by N-monomethyl-L-arginine (100 M) and oxypurinol (1 mM), respectively, was observed. NO synthase and xanthine oxidase activities were increased approximately three fold after treatment of synaptosomes with the gas phase of cigarette smoke as compared with the gas phase deprived of oxidants. Synaptosomes treated with the gas phase of cigarette smoke dramatically increased 3-nitrotyrosine production (used as an index of peroxynitrite formation). Synaptosomes treated with the gas phase of cigarette smoke, promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1,4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Gas phase deprived of oxidants showed a small but not statistically significant (p > 0.05) effect on both malondialdehyde and membrane fluidity. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells by oxidants contained in the gas phase of cigarette smoke lead to the formation of peroxynitrite a causative factor in neurotoxicity.     

Prior hydration of Brassica tournefortii seeds reduces the stimulatory effect of karrikinolide on germination and increases seed sensitivity to abscisic acid

Background and Aims

The smoke-derived compound karrikinolide (KAR₁) shows significant potential as a trigger for the synchronous germination of seeds in a variety of plant-management contexts, from weed seeds in paddocks, to native seeds when restoring degraded lands. Understanding how KAR₁ interacts with seed physiology is a necessary precursor to the development of the compound as an efficient and effective management tool. This study tested the ability of KAR₁ to stimulate germination of seeds of the global agronomic weed Brassica tournefortii, at different hydration states, to gain insight into how the timing of KAR₁ applications in the field should be managed relative to rain events.

Methods

Seeds of B. tournefortii were brought to five different hydration states [equilibrated at 15 % relative humidity (RH), 47 % RH, 96 % RH, fully imbibed, or re-dried to 15 % RH following maximum imbibition] then exposed to 1 nm or 1 µm KAR₁ for one of five durations (3 min, 1 h, 24 h, 14 d or no exposure).

Key Results

Dry seeds with no history of imbibition were the most sensitive to KAR₁; sensitivity was lower in seeds that were fully imbibed or fully imbibed then re-dried. In addition, reduced sensitivity to KAR₁ was associated with an increased sensitivity to exogenously applied abscisic acid (ABA).

Conclusions

Seed water content and history of imbibition were found to significantly influence whether seeds germinate in response to KAR₁. To optimize the germination response of seeds, KAR₁ should be applied to dry seeds, when sensitivity to ABA is minimized.     

Nicotine and apoptosis

Cigarette smoking is associated with a plethora of different diseases. Nicotine is the addictive component of cigarette but also acts onto cells of the non-neuronal system, including immune effector cells. Although nicotine itself is usually not referred to as a carcinogen, there is ongoing debate whether nicotine functions as a ‘tumor enhancer.' By binding to nicotinic acetylcholine receptors, nicotine deregulates essential biological processes like angiogenesis, apoptosis, and cell-mediated immunity. Apoptosis plays critical roles in a wide variety of physiologic processes during fetal development and in adult tissue and is also a fundamental aspect of the biology of malignant diseases. This review provides an overlook how nicotine influences apoptotic processes and is thus directly involved in the etiology of pathological conditions like cancer and obstructive diseases.     

2-Amino-9H-pyrido[2,3-b]indole (AαC) Adducts and Thiol Oxidation of Serum Albumin as Potential Biomarkers of Tobacco Smoke

2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine formed during the combustion of tobacco. AαC undergoes bioactivation to form electrophilic N-oxidized metabolites that react with DNA to form adducts, which can lead to mutations. Many genotoxicants and toxic electrophiles react with human serum albumin (albumin); however, the chemistry of reactivity of AαC with proteins has not been studied. The genotoxic metabolites, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC), 2-nitroso-9H-pyrido[2,3-b]indole (NO-AαC), N-acetyloxy-2-amino-9H-pyrido[2,3-b]indole (N-acetoxy-AαC), and their [¹³C₆]AαC-labeled homologues were reacted with albumin. Sites of adduction of AαC to albumin were identified by data-dependent scanning and targeted bottom-up proteomics approaches employing ion trap and Orbitrap MS. AαC-albumin adducts were formed at Cys³⁴, Tyr¹⁴⁰, and Tyr¹⁵⁰ residues when albumin was reacted with HONH-AαC or NO-AαC. Sulfenamide, sulfinamide, and sulfonamide adduct formation occurred at Cys³⁴ (AαC-Cys³⁴). N-Acetoxy-AαC also formed an adduct at Tyr³³². Albumin-AαC adducts were characterized in human plasma treated with N-oxidized metabolites of AαC and human hepatocytes exposed to AαC. High levels of N-(deoxyguanosin-8-yl)-AαC (dG-C8-AαC) DNA adducts were formed in hepatocytes. The Cys³⁴ was the sole amino acid of albumin to form adducts with AαC. Albumin also served as an antioxidant and scavenged reactive oxygen species generated by metabolites of AαC in hepatocytes; there was a strong decrease in reduced Cys³⁴, whereas the levels of Cys³⁴ sulfinic acid (Cys-SO₂H), Cys³⁴-sulfonic acid (Cys-SO₃H), and Met³²⁹ sulfoxide were greatly increased. Cys³⁴ adduction products and Cys-SO₂H, Cys-SO₃H, and Met³²⁹ sulfoxide may be potential biomarkers to assess exposure and oxidative stress associated with AαC and other arylamine toxicants present in tobacco smoke.