Periodontal cytokines profile under orthodontic force and extracorporeal shock wave stimuli in a rat model.

BACKGROUND AND OBJECTIVE: Extracorporeal shock wave therapy has been used in various clinical conditions as a result of its ability to stimulate healing processes in acute and chronic inflammatory states. Orthodontic force application triggers an inflammatory reaction in the periodontal tissue surrounding the involved teeth, resulting in tooth movement. Preliminary work revealed that extracorporeal shock wave therapy increased the expression of the inflammatory cytokines involved. Our aim was to investigate the expression of inflammatory cytokines in the periodontal tissues following orthodontic force induction, with and without shock wave therapy, in experimental rats. MATERIAL AND METHODS: An orthodontic appliance was fabricated and applied between the molars and the incisors of adult Wistar rats. In conjunction with orthodontic force commencement, the rats were treated with a single episode of 1000 shock waves. Every day, during the 3 d of the study, rats were killed and the immunolocalization of RANKL, interleukin (IL)-1ß, IL-6 and tumor necrosis factor-alpha was evaluated. RESULTS: The percentage of the area staining positively for all inflammatory cytokines during the first 2 d decreased statistically significantly more in the shock wave-treated group compared with the nontreated control group. On the first day, the percentage of the area staining positively for IL-1ß and RANKL on the compression side peaked in both groups, with a sequential rise in the number of TRAP-positive cells. CONCLUSION: The induction of shock wave therapy during orthodontic tooth movement influences the expression of different inflammatory cytokines in the tissue and might alter the expected periodontal remodeling rate.

Relationship between history of smoking, metabolic and inflammatory markers, parameters of body composition and muscle strength.

The objective of this study was to investigate the relationship between smoking history expressed by pack-years, metabolic and inflammatory markers, parameters of body composition (BC) and muscle strength among heavy smokers. A detailed smoking history was obtained from 49 heavy smokers (age = 44 ± 12, pack-years = 31 ± 23). Blood samples were analyzed for levels of glucose, lipids, liver enzymes and C-reactive protein (CRP). Anthropometric measurements included waist circumference and assessment of BC by dual energy X-ray absorptiometry (DEXA) and bioelectrical impedance analysis (BIA). Muscle strength was assessed by handgrip dynamometry and predicted one-repetition maximum (p1RM) tests. Positive correlations were found between pack-years of smoking, fasting glucose, alkaline phosphatase and CRP levels. Pack-years were also positively correlated with waist circumference, body mass index (BMI), whole-body and trunk fat mass measured by both DEXA and BIA. A negative correlation was found between pack-years of smoking and muscle strength measured by p1RM for the leg press exercise. After adjustment for age, sex and BMI, a positive correlation remained between pack-years of smoking and CRP levels. In conclusion, after controlling for possible confounders, smoking history was found to be positively associated with CRP levels among heavy smokers.

Time and dose effects of cigarette smoke and acrolein on protein carbonyl formation in HaCaT keratinocytes.

Cigarette smoke (CS) is an important environmental source of human exposure to a highly toxic and chemically active α,ß-unsaturated aldehyde: acrolein. It is capable of causing protein carbonylation and dysfunction, especially in oral tissues of smokers, constantly exposed to CS toxic constituents. The foremost damage is considered to be cumulative, but even a short exposure can be potentially harmful. The objectives of the current study were to examine the short time and dose effects of direct CS and acrolein exposure on intracellular protein carbonylation in epithelial cells. HaCaT-keratinocytes were exposed to different doses of acrolein and whole phase CS using a unique smoking simulator apparatus that mimics the exposure in smokers. The rate of intracellular protein carbonyl modification was examined 10-60 min after the exposure by Western blot. In addition, the effect of pre-incubation with a thiol scavenger N-acetylcysteine (NAC) was also assessed. We found that intracellular protein carbonyls increased as fast as 10 min after CS exposure and their concentration doubled after 20 min, with a slight elevation afterwards. Also, carbonyl levels increased gradually as CS and acrolein doses were elevated. Addition of 1 mM NAC neutralized part of the damage. We conclude that CS and acrolein intracellular protein carbonylation is dose- and time- dependent. Even a short time exposure to CS and its aldehydic constituents can be potentially harmful.

Green tea drinking improves erythrocytes and saliva oxidative status in the elderly.

We have previously shown that green tea (GT) drinking combined with vitamin E supplementation reduced plasma protein carbonyls and increased erythrocytes catalase activity in exercising healthy elderly. In the present study we set out to investigate the antioxidative effects of GT drinking in an aging population. We performed an interventional, crossover, controlled prospective trial with 35 healthy elderly subjects (mean age 67.3±4.8 years), supplemented with four daily placebo maltodextrin "tea-bags" for 12 weeks, followed by four 1.5 g daily GT bags for another 12 weeks. Data were obtained at baseline, at the end of the placebo period, and at the end of the GT intervention period. We found that GT did not alter erythrocyte catalase activity. However, it provided protection against 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis which declined by 10.2% (p<0.001). No changes were observed in saliva oral peroxidase enzymes. Nonetheless, saliva total antioxidant capacity increased by 42.0% (p<0.01). Plasma oxidative products, such as protein carbonyls, lipid peroxides and thiobarbituric acid reactive substances (TBARS) were stable throughout the intervention period. We conclude that four daily cups of GT are well tolerated in elderly free living subjects. Our results demonstrate that both erythrocyte resistances to oxidation and saliva antioxidant capacity are improved by GT drinking. The clinical implications of these oxidation modifications require further research.

Body composition in heavy smokers: comparison of segmental bioelectrical impedance analysis and dual-energy X-ray absorptiometry.

Smokers tend to have lower body mass index, on one hand, and increased abdominal obesity, on the other hand. Also, low levels of lean mass (LM) and bone mineral content (BMC) were found among older smokers compared with non-smokers. This altered body composition and its consequences raise the need for simple and reliable methods for assessment of body composition in smokers. This study aimed to compare body composition assessment by segmental bioelectrical impedance analysis (sBIA) with the reference method, dual energy X-ray absorptiometry (DEXA). Body composition was measured by sBIA (Tanita BC-545) and DEXA (Hologic) in 49 heavy smokers (>15 cigarettes/day, mean age 43.8±12.0). The comparison included correlations and differences between measurements obtained using the two methods as well as the Blande-Altman analysis. Whole-body fat mass (FM) and LM measured by the two methods were found to be highly correlated (r>0.9, p<0.001). Compared with DEXA, sBIA significantly overestimated whole-body LM and BMC (1,126 g and 382 g, respectively, p<0.01). The Bland-Altman analysis revealed a good agreement for whole-body FM and LM, but a poor agreement for BMC. The segmental FM percentage and LM were also highly correlated (r>0.9, p<0.001). However, sBIA significantly overestimated LM of the trunk and legs and underestimated the appendicular FM percentage. Verified by DEXA, sBIA provides reliable measures of whole-body LM, FM, and trunk FM in heavy smokers. A lesser degree of agreement was found for BMC, appendicular LM, and FM.

Peroxynitrite induces degradation of myosin heavy chain via p38 MAPK and muscle-specific E3 ubiquitin ligases in C2 skeletal myotubes.

Oxidative stress and inflammation play an important role in the catabolism of skeletal muscles. Recently, cigarette smoke (CS) was shown to stimulate muscle catabolism by activation of p38 MAPK and up-regulation of the muscle-specific E3 ubiquitin ligases (E3s) atrogin-1 and MuRF1 which are over-expressed during muscle atrophy. Peroxynitrite (ONOO-), an oxidative ingredient of CS, also produced during oxidative stress and inflammation, was previously shown to induce ubiquitination and degradation of muscle proteins. To investigate the involvement of p38 MAPK and the muscle-specific E3s in ONOO--induced muscle catabolism, C2 myotubes, differentiated from a myoblast cell line, were exposed to ONOO- (25 µM) in a time-dependent manner. Following exposure, degradation of myosin heavy chain (MyHC) and actin, activation of p38 MAPK, and levels of atrogin-1 and MuRF1 were studied by Western blotting. Peak phosphorylation of p38 MAPK was observed at 1 h of ONOO- exposure. ONOO- caused a significant increase in the levels of atrogin-1 and MuRF1. In accordance, a significant decrease in MyHC levels was observed in a time-dependent manner. These findings support previous studies in which the catabolic effects of ONOO- were shown. In addition, ONOO- was demonstrated to induce degradation of muscle proteins by activation of p38 MAPK and up-regulation of the muscle-specific E3s atrogin-1 and MuRF1.

The role of oxidation in FSL-1 induced signaling pathways of an atopic dermatitis model in HaCaT keratinocytes.

Oxidative stress (OS) is common in inflammatory conditions and may be important in atopic dermatitis (AD) etiology. The aim of this project was to study the involvement of oxidation in FSL-1 (deacylated lipoprotein)-triggered signaling pathways leading to AD-typical cytokine expression in HaCaT keratinocytes. HaCaT keratinocytes, pretreated with the inhibitor to OS N-acetylcysteine (NAC), were exposed to FSL-1, a stimulator of AD-related cytokines. Cytokines expression was studied by real time polymerase chain reaction (PCR); nuclear factor-kappa B (NF-κB) and p38 mitogen activated protein kinase (MAPK) activities were studied by western blotting; and the oxidative state of cells was determined by the dichlorofluorescein (DCF) assay. We found that endogenous OS in keratinocytes appeared 4 h after FSL-1 administration. OS activated NF-κB, but not p38 MAPK, and the inhibition of OS reduced FSL-1 induced interleukin (IL) 33, thymic stromal lymphopoietin (TSLP) and TNFα mRNA expression. We conclude that FSL-1 triggers an OS reaction in HaCaT keratinocytes, which is probably a secondary event affecting the expression of specific AD typical cytokines, possibly through the NF-κB pathways. This role of OS in the inflammatory response in AD is worth further investigating.

Essential amino acid leucine and proteasome inhibitor MG132 attenuate cigarette smoke induced catabolism in C2 myotubes.

Exposure to cigarette smoke (CS) and cigarette smoking have been shown to promote catabolism of skeletal muscle. Previous studies and recent findings from our laboratory have demonstrated the involvement of the ubiquitin proteasome system and the muscle-specific E3 ubiquitin ligases MAFbx/atrogin-1 and MuRF1 in CS induced skeletal muscle catabolism. The essential amino acid leucine is a known anticatabolic agent that improves skeletal muscle metabolism in various atrophic conditions. To examine the protective effect of leucine and proteasome inhibition in CS induced muscle catabolism, C2 myotubes, from an in vitro skeletal muscle cell line, were exposed to CS in the presence or absence of leucine and a proteasome inhibitor, MG132. Diameter of myotubes, levels of the main contractile proteins - myosin heavy chain and actin, expression of MAFbx/atrogin-1 and MuRF1 were studied by microscopy, Western blotting, and qPCR. Leucine pretreatment prevented the CS-induced reduction in diameter of myotubes and degradation of myosin heavy chain by suppressing the upregulation of MAFbx/atrogin-1 and MuRF1. MG132 also attenuated the CS-induced decrease in diameter of myotubes and degradation of myosin heavy chain. Our findings demonstrate that supplementation with the essential amino acid leucine and inhibition of the proteasome may protect skeletal muscle from CS induced catabolism.

Effects of cigarette smoke on salivary protein tyrosine nitration.

INTRODUCTION: Nitration of tyrosine and tyrosine-containing proteins and their roles in pathophysiology have just recently been reviewed. Despite low yields of tyrosine modifications, nitration of tyrosine residues may inactivate important proteins. Nitrotyrosine can be formed by various nitrating agents, including peroxynitrite. Thus, the occurrence of nitrotyrosine-containing proteins in vivo should be regarded as a general indication of tissue damage induced by reactive nitrogen species such as peroxynitrite. This strongly suggests that peroxynitrite could be formed in vivo under certain pathophysiological conditions. OBJECTIVE: Our aim in this study was to elucidate the effect of cigarette smoke (CS) on nitrotyrosine formation in saliva proteins. METHODS: We exposed saliva to CS, in vitro, and used Western Blotting (WB) and monoclonal anti-nitrotyrosine antibody to assess the level of saliva protein nitration. RESULTS: As saliva contains extensive amounts of nitrites, it was no surprise that at basal levels, saliva proteins, albumin, and α-amylase all were already nitrated. The WB also revealed that with continuous exposure to CS the tyrosine nitration of both albumin and α-amylase is declining significantly after 3 h. A quite similar effect was seen after exposure to aldehydes, but to a less extent as compared to CS. Exposure of nitrotyrosine-modified bovine serum albumin (BSA-N) to aldehydes, produced a similar effect, meaning a decrease in tyrosine nitration. CONCLUSIONS: These findings might be explained by the possible ability of CS aldehydes to reduce protein-bound nitro group to an amine. Another proposed mechanism is that CS unsaturated aldehydes react with proteins mainly through Michael addition reaction; leading to the generation of stable aldehyde-protein adducts (APA). Thus, it may react with nitro groups of saliva proteins, like albumin or α-amylase, to generate APA, which ultimately, may not be recognized by our antibody. Another possible mechanism, is interaction between the aldehyde group with the hydroxyl group of the 3-nitrotyrosine, forming a hemiacetal, which is not recognized by the antibody. This mechanism might explain the difference in the denitration effects caused by the saturated aldehyde acetaldehyde, which exists in large amounts in CS, and unsaturated aldehydes. Therefore, it is possible that the main player in the CS smoke denitration effect on salivary proteins is the aldehyde group and not the double bond of unsaturated aldehydes.

Acute incremental exercise to maximal performance does not cause alterations in serum oxidant levels of healthy young individuals.

AIM: This study was designed to analyze serum oxidative stress (OS) levels in healthy young individuals performing a routine maximal aerobic exercise and to evaluate the correlation between OS levels and physiological parameters. METHODS: Serum OS levels were studied by thermochemiluminescence (TCL) parameters at rest and following maximal aerobic exercise in 85 healthy young subjects. Levels were measured by a real time on line TCL assay (higher TCL-Ratio and TCL-H3 = lower OS level). RESULTS: Aerobic capacity had no effect on baseline OS levels. Post-exercise OS levels correlated with maximal oxygen uptake (V.O(2max)) (P<0.005), delta V.O(2) (V.O(2max)- V.O(2)rest) (P<0.005), anaerobic threshold (VTH) (P<0.01), and total oxygen uptake (especially O(2) after VTH), (P<0.005). TCL-Ratio was related to total running time (P<0.01), as well. Post-exercise OS levels for the whole study group did not vary from baseline values. However, individuals with higher fitness level (V.O(2max) >percentile 60) had significantly lower values of TCL-H3 (P=0.04) and tended to have lower TCL-Ratio, indicating they had elevated OS levels. In a multivariate analysis OS level was most affected by V.O(2) after VTH (anaerobic phase of the test) (P=0.003; adjusted odds ratio of 3.41, 95% confidence interval: 1.55-7.48). CONCLUSIONS: In conclusion, acute incremental exercise to maximal performance does not cause alterations in serum oxidant levels of healthy young individuals. In healthy individuals performing maximal aerobic exercise, OS levels correlate with maximal aerobic power.

Increased pro-inflammatory activity and impairment of human monocyte differentiation induced by in vitro exposure to cigarette smoke.

Cigarette smoking (CS) is associated with a variety of human pathologies including cardiovascular disease and cancer. Human monocytes are prevalent in oral and respiratory mucosa and may be affected by exposure to CS, which induces oxidative stress. As a result, up-regulation of nuclear factor-kappaB (NF-kappaB) may occur. Our aims were to analyze a possible regulatory effect of CS on NF-kappaB activity in human monocytes. Human monocyte cell lines were exposed to CS in vitro. Our findings show that in vitro exposure to CS did not affect viability of human monocytes and was associated with increased production and secretion of IL-8 and up-regulation of certain C-C chemokines. Inhibition of NF-kappaB with curcumin or parthenolide resulted in a decrease of IL-8 secretion. CS also impaired the differentiation of monocytes. However, induced secretion of IL-8 from differentiated monocytes was not impaired. Our results indicate that exposure to CS stimulates pro-inflammatory activity of human monocytes through the activation of NF-kappaB pathway and also interferes with monocyte differentiation, which could play a role in the carcinogenic effects of cigarette smoking.

Effects of cigarette smoke borne reactive nitrogen species on salivary alpha-amylase activity and protein modifications.

Cigarette smoke (CS) is associated with a variety of human pathologies including cardiovascular disease and cancer. Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck. The major inducer of OSCC is exposure to tobacco. Recent studies demonstrated that oxidative and nitrosative stress contributes to the development of oral carcinogenesis through DNA damage. All salivary reactive nitrogen species (RNS) analyzed from OSCC patients are significantly higher in comparison with healthy subjects. Our findings show that CS and external RNS addition induced reduction in alpha-amylase activity and produced some excited carbonyl formation, but to a much less extant than CS. The addition of epigallocatechine-3-gallate (EGCG) to saliva produced no protective effect against damage to alpha-amylase activity. Our proposed mechanism for the decrease in alpha-amylase activity is the formation of adducts at SH groups of the alpha-amylase active site. In this case, EGCG was unable to counteract this phenomenon, as it does not reduce the concentration of disulfides, and does not alter the amount of protein-SH moieties. However, EGCG did reduce the levels of excited carbonyl formation. Our results indicate that although RNS are abundant in CS, a significant decrease in amylase activity is due to other components in CS, probably aldehydes, reacting with the thiol group of proteins by the Michael addition reaction.

Evidence-based roads to the promotion of health in old age.

The increase in life expectancy, along with the accompanying ongoing increase in the proportion and absolute numbers of nonagenarians and centenarians have set forth the curiosity regarding the question of the quality of health in very old age. Studies on that issue have pointed to the fact that the very old people are actually healthier than originally predicted on the basis of the earlier studies on aging. Current efforts are thus invested in elucidating the possible basis of health in the very old people, as well as better understanding of potential causes of frailty and common diseases in old age. This review recounts on the various aspects underlying evidence-based recommendations for healthy life in old age. We focus on the genetic and non-genetic bases of aging and longevity, and the various directions towards the promotion of health, both via avoiding, or eliminating risk factors and deleterious effects, as well as conducting healthy lifestyle - in terms of proper nutrition and physical exercise. Next, we touch upon preventive medicine, particularly as related to vaccination, with a note also on the need for a reasonable use of medications. In addition, we report about the developing area of regenerative medicine and its potential in relation to the prevention of damage and possible strategies towards tissue repair in cases of age-related degenerative processes.

Exposure to pro-inflammatory cytokines upregulates MMP-9 synthesis by mesenchymal stem cells-derived osteoprogenitors.

An intimate interplay exists between the bone and the immune system, which has been recently termed osteoimmunology. The activity of immune cells affects the intrinsic balance of bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts. The aim of this study was to determine the possible interaction between inflammatory-induced conditions and matrix metalloproteinases-2,-9 (MMP-2,-9) synthesis and secretion by bone marrow-derived osteoprogenitor cells during advanced stages of osteogenesis. Rat bone marrow-derived mesenchymal stem cells (MSCs) were cultured in the presence of osteogenic supplements in order to direct the cells towards the osteogenic differentiation lineage. At the late stages of osteogenesis, assessed by histochemistry, immunohistochemistry and RT-PCR, cultures were exposed to pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha). Biochemical, histochemical and molecular biology techniques were used to discern the influence of pro-inflammatory cytokines on MMP-2,-9 synthesis and secretion. Results indicated that MMP-9 synthesis and secretion were significantly induced after exposure to the cytokines (TNF-alpha, IL-1 alpha) treatment, while MMP-2 levels remained unchanged. These results indicate that in response to inflammatory processes, osteoblasts, in addition to osteoclasts, can also be involved and contribute to the process of active bone resorption by secretion and activation of MMPs.

The biological basis of the bone-muscle inter-relationship in the algorithm of fracture healing.

The biological cascade of fracture healing is intimately linked to the muscle envelope. It further depends on the preservation of stable, perpetual axial micromovements. The current study was designed to demonstrate that high molecular weight bioactive substances diffuse from the muscle envelope to initiate osteoinductive activity at experimental fracture sites. Forty-eight rats underwent an experimental fracture of the left tibia and stabilization with an intramedullary 20-gauge needle. The animals were divided into 4 groups (A-D) of 12 rats each according to the post-fracture treatment. In group A (control) no additional treatment was applied following fracture and intramedullary fixation. In groups B, C, and D, a nitrocellulose membrane of various sizes was wrapped around the fracture, separating the periosteum from the muscle envelope. The groups differed by the membrane pore size, allowing passage of the following molecular sizes: 50 kilodaltons (kDa), 12 to 14 kDa, and 3.5 kDa in groups B, C, and D, respectively. Four animals in each group were sacrificed 2, 5, and 10 weeks after the procedure for radiographic and histological evaluation of fracture healing. Radiographic evaluation revealed a decreased rate of bone synthesis that correlated with the nitrocellulose pore size. Morphological and functional analysis of the bone explants indicated poorly healed fractures in groups B, C, and D. Direct contact between fractured bone and its muscle envelope is essential for the biological sequence of new bone formation. The extent of obstruction between the fracture and its muscle envelope correlates with the delay in fracture healing.

Inhibition of salivary amylase activity by cigarette smoke aldehydes.

Cigarette smoke (CS) is a leading known cause of cancer and cardiovascular diseases worldwide. The mechanisms by which CS produces its damaging effects seem to be multifactorial. Among others, CS toxicity is due also to several compounds like alpha,beta-unsaturated aldehydes (acrolein, crotonaldehyde) and saturated aldehydes (acetaldehyde). Aldehydes could interact with thiol compounds of salivary proteins, leading to structural and functional alterations of these molecules. Prior in vitro studies have shown that there is a significant decrease in several known enzymatic activities following exposure to CS. Additionally, it was found that glutathione (GSH) has protective effect against the damaging role of CS to salivary enzymes, emphasizing the role of thiol groups in the mechanism of inactivation of these enzymes. In this study, salivary amylase activity showed a significant inhibition following exposure to CS, and to external addition of purified aldehydes known to be present in CS, due probably to the interaction between aldehydes and -SH groups of the enzyme. Our results indicate that although saturated aldehydes are the chief aldehydes present in CS, a significant decrease in amylase activity was due to unsaturated aldehydes, reacting, probably, through their double bond with the thiol group of proteins by the Michael addition reaction.

Cigarette smoke-induced NF-kappaB activation in human lymphocytes: the effect of low and high exposure to gas phase of cigarette smoke.

Cigarette smoking is linked to various human disorders. Active and passive smokers suffer from inflammatory diseases of lungs and airways. Smoking-dependent airway inflammation is related to the cytotoxic effects of cigarette smoke (CS) and chronic recruitment of neutrophils and macrophages. NF-kappaB is a key inflammatory, redox-sensitive transcription factor. Its role in CS-induced airway inflammation is unclear. This study investigated CS-induced NF-kappaB activation in human lymphocytes and the possible involvement of oxidative insult in this activation. A method for accurate and reproducible exposure of lymphocytes to CS was developed. The intensity of CS exposure was linearly correlated with nitrite concentration originating from reactive oxygen species in CS. Mild, but not high exposure to CS, induced NF-kappaB in lymphocytes through the increase in oxidative stress and the reduction in the intracellular glutathione levels. These findings may have implications to active as well as to passive smokers, suffering from inflammatory diseases of lungs and airways.

Mechanisms underlying cigarette smoke-induced NF-kappaB activation in human lymphocytes: the role of reactive nitrogen species.

Cigarette smoke (CS) is an important source of reactive nitrogen species (RNS). It has been demonstrated that CS constitutes the highest source of exogenous nitric oxide and peroxynitrite to which humans are exposed. NF-kappaB is a key inflammatory, redox-sensitive transcription factor, which role in CS-induced airway inflammation is unclear. Moreover, the role of RNS in the activation of NF-kappaB and in inflammation has remained vague. This study investigated CS-induced NF-kappaB activation in human lymphocytes and assessed the involvement of CS-derived RNS in NF-kappaB activation and their possible biological effects. Mild, but not high, exposure to CS induced NF-kappaB in lymphocytes through IKK activation, I-kappaBalpha degradation, and the reduction in the intracellular glutathione levels. Peroxynitrite, but not NO, mimicked the effects of CS on NF-kappaB. Reduction of intracellular peroxynitrite formation by the inhibition of the mitochondrial respiratory chain resulted in decreased activation of NF-kappaB by CS. NF-kappaB-induced iNOS levels were increased in response to CS. Low levels of CS exposure induced classical NF-kappaB activation pathway in lymphocytes via intracellular formation of peroxynitrite, through a reaction between smoke-derived NO and endogenously produced superoxide. This NF-kappaB activation resulted in inflammatory gene expression, which may contribute to CS-related airway inflammation.

The role of reactive nitrogen species and cigarette smoke in activation of transcription factor NF-kappaB and implication to inflammatory processes.

Using the electromobility shift assay (EMSA) in the rat myoblast system, the activation of transcription factor NF-kappaB by reactive nitrogen species was evaluated. Two distinct patterns of activation were demonstrated. Whereas NO donor, SNAP, activated NF-kappaB in the classical pathway, which led to a transient response, NF-kappaB activation by peroxynitrite donor, SIN-1, was mediated by an alternative pathway, which has been demonstrated in previous works to involve tyrosine nitration of the NF-kappaB inhibitory protein I-kappaB alpha. This led to a constitutive non-transient activation of NF-kappaB and a prolonged inflammatory reaction. Lymphocytes exposed to mild intensity of cigarette smoke for 8 h, which activated NF-kappaB, exhibited a decrease in the fraction of apoptotic cells from 27% to 19% compared with lymphocytes exposed to atmospheric air, using the FACS Annexin V assay. This also has been shown in previous works to be mediated by peroxynitrite. Thus, mild exposure to cigarette smoke induces NF-kappaB activation, which can attenuate apoptosis in human lymphocytes and lead to prolonged inflammatory response. A possible proposed mechanism for induction of chronic inflammatory response may involve peroxynitrite-induced activation of NF-kappaB.

Saliva--a pivotal player in the pathogenesis of oropharyngeal cancer.

Oropharyngeal (OP) cancer, which is usually squamous cell carcinoma, is the most common head and neck malignancy and accounts for 2-4% of all new cancers. It is primarily induced by exposure to tobacco. The paradigm of cigarette smoke (CS)-induced OP cancer's pathogenesis is based on the assumption that a constant direct attack of various CS carcinogens causes widespread accumulating cellular and DNA aberrations in the OP mucosal cells, in turn eventually resulting in malignant transformation. However, there is never a direct contact between CS and the OP mucosa. Saliva, bathing the mucosa from the oral cavity to the larynx, always intervenes, and CS must first interact with saliva before it reaches the mucosa. The current study investigated the role of saliva in the pathogenesis of OP cancer. A synergistic effect of CS and saliva on oral cancer cells was demonstrated. This synergism is based on the reaction between redox active metals in saliva and low reactive free radicals in CS, which results in the production of highly active hydroxyl free radicals. Thus, when exposed to CS, salivary behavior is reversed and the saliva loses its antioxidant capacity and becomes a potent pro-oxidant milieu. The devastating role of CS-borne aldehydes was demonstrated as well. Based on these results and on our recent reports demonstrating that CS destroys various salivary components, including protective ones such as peroxidase, the most important salivary antioxidant enzyme, a comprehensive view of the pivotal role of saliva in the pathogenesis of CS-induced OP cancer is suggested.