Modulation of smoke-induced DNA and microRNA alterations in mouse lung by licofelone, a triple COX-1, COX-2 and 5-LOX inhibitor

Chronic inflammation plays a crucial role in the carcinogenesis process and in particular in smoking-related carcinogenesis. Therefore, anti-inflammatory agents provide an interesting perspective in the prevention of smoking-associated cancers. Among nonsteroidal anti-inflammatory drugs (NSAIDs), licofelone is a triple inhibitor of both cyclooxygenases (COX-1 and COX-2) and of 5-lipooxygenase (5-LOX) that has shown some encouraging results in cancer prevention models. We previously showed that the dietary administration of licofelone, starting after weanling, to Swiss H mice exposed for 4 months to mainstream cigarette smoke since birth attenuated preneoplastic lesions of inflammatory nature in both lung and urinary tract, and had some effects on the yield of lung tumors at 7.5 months of age. The present study aimed at evaluating the early modulation by licofelone of pulmonary DNA and RNA alterations either in smoke-free or smoke-exposed H mice after 10 weeks of exposure. Licofelone protected the mice from the smoke-induced loss of body weight and significantly attenuated smoke-induced nucleotide alterations by decreasing the levels of bulky DNA adducts and 8-hydroxy-2'-deoxyguanosine in mouse lung. Moreover, the drug counteracted dysregulation by smoke of several pulmonary microRNAs involved in stress response, inflammation, apoptosis, and oncogene suppression. However, even in smoke-free mice administration of the drug had significant effects on a broad panel of microRNAs and, as assessed in a subset of mice used in a parallel cancer chemoprevention study, licofelone even enhanced the smoke-induced systemic genotoxic damage after 4 months of exposure. Therefore, caution should be paid when administering licofelone to smokers for long periods

Mechanisms Of Inhibition Of Cigarette Smoke Genotoxicity And Carcinogenicity

Epidemiological studies have demonstrated that it is possible to prevent lung cancer and other smoke-related diseases by avoiding exposures to tobacco smoke. A complementary strategy is chemoprevention, which is based on the administration of dietary and pharmacological agents, which is addressed to (a) addicted active smokers, who are unable to quit smoking, (b) ex-smokers, who are still at risk for several years, and (c) involuntary smokers, including passively exposed individuals as well as transplacentally exposed individuals. The biological effects of cigarette smoke (CS) as a complex mixture, either mainstream (MCS) or sidestream (SCS) or environmental (ECS), have been poorly explored. We showed that MCS and ECS induce a broad variety of alterations of intermediate biomarkers in animal models, including adducts to nuclear DNA and mtDNA, oxidatively generated DNA damage, proliferation, apoptosis, alterations of oncogenes and tumor suppressor genes, multigene expression, microRNA and proteome profiles as well as cytogenetic damage in the respiratory tract, bone marrow and peripheral blood. CS-altered end-points were variously modulated by chemopreventive agents of natural or pharmacological origin, such as N-acetyl-L-cysteine (NAC), 1,2-dithiole-3-thione, oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate (PEITC), indole-3-carbinol, sulindac, and budesonide. Combinations of agents were also assayed. Since it is difficult to assess the efficacy of chemopreventives in clinical trials, it is essential to understand the mechanisms by which certain agents are expected to prevent smoke-related cancer. Preclinical studies are also useful to demonstrate the potential efficacy of chemopreventive agents. Unfortunately, until recently a suitable animal model for evaluating CS carcinogenicity and its chemoprevention was not available. We demonstrated that ECS and especially MCS become potently carcinogenic when exposure of mice starts at birth, as shown by very short latency times, high incidence and multiplicity of benign lung tumors, early occurrence of malignant lung tumors, and lesions in other organs. This mouse model was successfully used to demonstrate the ability of NAC, PEITC, and budesonide to prevent smoke-induced lung cancer, according to protocols mimicking the situation either in current smokers or in ex-smokers. Other dietary or pharmacological agents, including curcumin, anthocyanins, myo-inositol, SAHA, bexarotene and pioglitazone, are now under study. NAC was even successful to prevent lung cancer induced by MCS after birth when it was administered during the prenatal life. Therefore, it is now possible to investigate in vivo not only alterations of intermediate biomarkers but also the modulation of CS carcinogenesis by chemopreventive agents working with different mechanisms

Modulation of genomic and epigenetic end-points by celecoxib

Celecoxib, a nonsteroidal anti-inflammatory drug that selectively targets cyclooxygenase-2, is a promising cancer chemopreventive agent. However, safety concerns have been raised in clinical trials evaluating its ability to prevent colorectal adenomas. The rationale for the herein reported studies was to analyze genomic and epigenetic end-points aimed at investigating both the chemopreventive properties of celecoxib towards cigarette smoke-associated molecular alterations and its possible adverse effects. We carried out three consecutive studies in mice treated with either smoke and/or celecoxib. Study 1 investigated early DNA alterations (DNA adducts, oxidative DNA damage, and systemic genotoxic damage) and epigenetic alterations (expression of 1,135 microRNAs) in lung and blood of Swiss H mice; Study 2 evaluated the formation of DNA adducts in lung, liver, and heart; and Study 3 evaluated the expression of microRNAs in 10 organs and 3 body fluids of ICR (CD-1) mice. Surprisingly, the oral administration of celecoxib to smoke-free mice resulted in the formation of DNA adducts in both lung and heart and in dysregulation of microRNAs in mouse organs and body fluids. On the other hand, celecoxib attenuated smoke-related DNA damage and dysregulation of microRNA expression. In conclusion, celecoxib showed pleiotropic properties and multiple mechanisms by counteracting the molecular damage produced by smoke in a variety of organs and body fluids. However, administration of celecoxib to non-smoking mice resulted in evident molecular alterations, also including DNA and RNA alterations in the heart, which may bear relevance in the pathogenesis of the cardiovascular adverse effects of this drug

Release of MicroRNAs into body fluids from ten organs of mice exposed to cigarette smoke

Purpose: MicroRNAs are small non-coding RNAs that regulate gene expression, thereby playing a role in a variety of physiological and pathophysiological states. Exposure to cigarette smoke extensively downregulates microRNA expression in pulmonary cells of mice, rats, and humans. Cellular microRNAs are released into body fluids, but a poor parallelism was previously observed between lung microRNAs and circulating microRNAs. The purpose of the present study was to validate the application of this epigenetic biomarker by using less invasive collection procedures. Experimental design: Using microarray analyses, we measured 1135 microRNAs in 10 organs and 3 body fluids of mice that were either unexposed or exposed to mainstream cigarette smoke for up to 8 weeks. The results obtained with selected miRNAs were validated by qPCR. Results: The lung was the main target affected by smoke (190 dysregulated miRNAs), followed by skeletal muscle (180), liver (138), blood serum (109), kidney (96), spleen (89), stomach (36), heart (33), bronchoalveolar lavage fluid (32), urine (27), urinary bladder (12), colon (5), and brain (0). Skeletal muscle, kidney, and lung were the most important sources of smoke-altered microRNAs in blood serum, urine, and bronchoalveolar lavage fluid, respectively. Conclusions: microRNA expression analysis was able to identify target organs after just 8 weeks of exposure to smoke, well before the occurrence of any detectable histopathological alteration. The present translational study validates the use of body fluid microRNAs as biomarkers applicable to human biomonitoring for mechanistic studies, diagnostic purposes, preventive medicine, and therapeutic strategies

Upalni i hematotoksični potencijal metabolita plijesni Stachybotrys chartarum (Ehrenb.) Hughes u zatvorenim prostorijama

Mould Stachybotrys chartarum (Ehrenb.) Hughes is known to pose a health risk in indoor environments. Most of its strains can produce several intra- and extracellular trichothecene mycotoxins. Complex secondary metabolites of stachybotrys isolates from mouldy dwellings/public buildings in Slovakia were intratracheally instilled in Wistar male rats (4 μg in 0.2 mL of 0.2 % dimethylsulphoxide; diacetoxyscirpenol as the positive control). After three days, haematological parameters were measured in peripheral blood and infl ammatory response biomarkers in bronchoalveolar lavage fl uid (BALF), and the results were statistically analysed. Exometabolites proved to suppress red blood cell (RBC), decreasing the total RBC count, haemoglobin, and haematocrit. The exposed rats showed signifi cantly higher total BALF cell count, indicating infl ammation, lower alveolar macrophage counts, and increased granulocyte count related to the BALF cells. Due to haematotoxic and infl ammation-inducing properties, metabolites of S. chartarum can cause damage to the airways and haematological disorders in occupants of mouldy buildings.Plijesan Stachybotrys chartarum (Ehrenb.) Hughes poznata je kao rizični mikroorganizam u zatvorenim prostorijama. Većina njezinih sojeva može proizvesti nekoliko unutarstaničnih i izvanstaničnih trikotecenskih mikotoksina. Muškim Wistar štakorima instilirani su intratrahealno kompleksni sekundarni metaboliti stahibotrisa izolirani iz stambenih i javnih zgrada u Slovačkoj zahvaćenima plijesni (4 μg na 0,2 mL 0,2 %-tnog dimetilsulfoksida; dok se diacetoksiscirpenol rabio kao pozitivna kontrola). Tri dana kasnije izmjereni su hematološki parametri u perifernoj krvi te biopokazatelji upalnoga odgovora u bronhoalveolarnome ispirku te su rezultati obrađeni statistički. Pokazalo se da egzometaboliti suprimiraju eritrocite, smanjujući njihov ukupni broj, hemoglobin i hematokrit. Izloženi štakori imali su značajno veći broj stanica u bronhoalveolarnome ispirku, što upućuje na upalu, dok im je broj alveolarnih makrofaga bio manji, a broj granulocita povezanih sa stanicama u ispirku veći. Zbog svojih hematotoksičnih i upalnih svojstava S. chartarum može dovesti do oštećenja dišnih putova i poremećaja u krvotvornome sustavu osoba koje žive i/ili rade u zgradama zahvaćenima tom plijesni

Comparative Functional Genomics Analysis of NNK Tobacco-Carcinogen Induced Lung Adenocarcinoma Development in Gprc5a-Knockout Mice

Background: Improved understanding of lung cancer development and progression, including insights from studies of animal models, are needed to combat this fatal disease. Previously, we found that mice with a knockout (KO) of G-protein coupled receptor 5A (Gprc5a) develop lung tumors after a long latent period (12 to 24 months). Methodology/Principal Findings: To determine whether a tobacco carcinogen will enhance tumorigenesis in this model, we administered 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) i.p. to 2-months old Gprc5a-KO mice and sacrificed groups (n = 5) of mice at 6, 9, 12, and 18 months later. Compared to control Gprc5a-KO mice, NNK-treated mice developed lung tumors at least 6 months earlier, exhibited 2- to 4-fold increased tumor incidence and multiplicity, and showed a dramatic increase in lesion size. A gene expression signature, NNK-ADC, of differentially expressed genes derived by transcriptome analysis of epithelial cell lines from normal lungs of Gprc5a-KO mice and from NNK-induced adenocarcinoma was highly similar to differential expression patterns observed between normal and tumorigenic human lung cells. The NNK-ADC expression signature also separated both mouse and human adenocarcinomas from adjacent normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of Ube2c, Mcm2, and Fen1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively

Antioxidant intervention of smoking-induced lung tumor in mice by vitamin E and quercetin

<p>Abstract</p> <p>Background</p> <p>Epidemiological and in vitro studies suggest that antioxidants such as quercetin and vitamin E (VE) can prevent lung tumor caused by smoking; however, there is limited evidence from animal studies.</p> <p>Methods</p> <p>In the present study, Swiss mouse was used to examine the potential of quercetin and VE for prevention lung tumor induced by smoking.</p> <p>Results</p> <p>Our results suggest that the incidence of lung tumor and tumor multiplicity were 43.5% and 1.00 ± 0.29 in smoking group; Quercetin has limited effects on lung tumor prevention in this in vivo model, as measured by assays for free radical scavenging, reduction of smoke-induced DNA damage and inhibition of apoptosis. On the other hand, vitamin E drastically decreased the incidence of lung tumor and tumor multiplicity which were 17.0% and 0.32 ± 0.16, respectively (p < 0.05); and demonstrated prominent antioxidant effects, reduction of DNA damage and decreased cell apoptosis (p < 0.05). Combined treatment with quercetin and VE in this animal model did not demonstrate any effect greater than that due to vitamin E alone. In addition, gender differences in the occurrence of smoke induced-lung tumor and antioxidant intervention were also observed.</p> <p>Conclusion</p> <p>We conclude that VE might prevent lung tumor induced by smoking in Swiss mice.</p