Unburned Tobacco Cigarette Smoke Alters Rat Ultrastructural Lung Airways and DNA.

INTRODUCTION: Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. METHODS: Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. RESULTS: Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. CONCLUSIONS: Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. IMPLICATIONS: IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model.

The Mediterranean Athlete's Nutrition: Are Protein Supplements Necessary?

(1) Background: It is recommended that an athlete, in order to ensure correct nutrition and performance, should consume between 1.2 and 2.0 g/kg/day of protein, while the daily recommended protein intake for a non-athlete is 0.8and 0.9 mg/kg/day. It is unclear if athletes living in Mediterranean countries are able to meet protein requirements without supplementation, since Mediterranean diet de-emphasizes meat and meat products. (2) Methods: 166 athletes (125 males) enrolled between 2017 and 2019 were required to keep a dietary journal for three consecutive days (2 workdays and 1 weekend day). Athletes had to be >18 years old, train in a particular sport activity more than 3 h a week and compete at least at an amateur level. Journal data were collected and then translated into macro-nutrient content (grams of protein, carbohydrates, and lipids) by a nutritionist. (3) Results: The protein intake reported by this specific population vary slightly from the Academy of Nutrition and Dietetics (AND), Dietitians of Canada (DC), and the American College of Sports Medicine (ACSM) joint statement recommendation level. Average protein levels without protein supplementation fell within the protein guidelines. Counterintuitively, the intake among those who supplemented their diet with protein was higher compared with those who did not, even when excluding the contribution of supplements. Although the majority of subjects participating in the study were able to meet protein intake recommended for athletes without protein supplementation, 27% of athletes were below the guideline range. (4) Conclusions: these data suggest that athletes' nutrition should be more often evaluated by a nutritionist and that they will benefit from increasing their nutritional knowledge in order to make better food choices, resorting to protein supplementation only when effectively needed.

Development and Longevity: Cellular and Molecular Determinants - A Mini-Review.

Across species, development and longevity are tightly linked. We discuss the relevant literature and suggest that the root for this stringent relationship is the rate of development. The basis for the relationship between rate of development and longevity lies in adaptations that have occurred through evolution at multiple levels of biological complexity: organism, organ, cellular, and molecular. Thus, the analysis of the relationship is of interest for multiple fields of biology.

A pro longevity role for cellular senescence.

Cellular senescence is a fundamental process that may play positive or detrimental roles for the organism. It is involved in tissue development and in tumor prevention although during aging is becoming a detrimental process contributing to the decline of tissue functions. In previous investigations, we have uncovered a better capacity to detect DNA damage in cells from long-lived mammals. Here, we report that cultured cells derived from long-lived species have a higher propensity to undergo senescence when challenged with DNA damage than cells derived from short-lived species. Using a panel of cells derived from six mammals, which range in lifespan from 3-4 years up to 120 years, we examined cell cycle response, induction of apoptosis and of cellular senescence. All species exhibited a cell cycle arrest while induction of apoptosis was variable. However, a significant positive correlation was found between the relative percent of cells, within a population which entered senescence following damage, and the lifespan of the species. We suggest that cellular senescence may have a positive role during development allowing it to contribute to the evolution of longevity.

Co-carcinogenic effects of vitamin E in prostate.

A large number of basic researches and observational studies suggested the cancer preventive activity of vitamin E, but large-scale human intervention trials have yielded disappointing results and actually showed a higher incidence of prostate cancer although the mechanisms underlying the increased risk remain largely unknown. Here we show through in vitro and in vivo studies that vitamin E produces a marked inductive effect on carcinogen-bioactivating enzymes and a pro-oxidant status promoting both DNA damage and cell transformation frequency. First, we found that vitamin E in the human prostate epithelial RWPE-1 cell line has the remarkable ability to upregulate the expression of various phase-I activating cytochrome P450 (CYP) enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), giving rise to supraphysiological levels of reactive oxygen species. Furthermore, our rat model confirmed that vitamin E in the prostate has a powerful booster effect on CYP enzymes associated with the generation of oxidative stress, thereby favoring lipid-derived electrophile spread that covalently modifies proteins. We show that vitamin E not only causes DNA damage but also promotes cell transformation frequency induced by the PAH-prototype benzo[a]pyrene. Our findings might explain why dietary supplementation with vitamin E increases the prostate cancer risk among healthy men.

Complete Disaggregation of MCF-7-derived Breast Tumour Spheroids with Very Low Concentrations of α-Mangostin Loaded in CD44 Thioaptamer-tagged Nanoparticles.

Background: α-Mangostin (αMG) is a natural substance that exerts a wide range of antitumor effects. Recently, we described that free αMG was able to dissociate multicellular tumour spheroids (MCTSs) generated from breast carcinoma cells and to reduce their cellular viability and motility. Here, αMG was encapsulated into lipidic nanoparticles (NPs), conjugated or not to a CD44 thioaptamer, and the anticancer action evaluated against MCF-7 breast MCTSs. Methods: NPs containing αMG were formulated with a core of polylactic-co-glycolyc acid. Some of them were decorated with a CD44 thioaptamer using as catalysts 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Both size and density of MCF-7-derived MCTSs were monitored during 72 h of treatment with NPs carrying 0.1, 0.5 and 1.0 µg/ml final concentrations of αMG. MCTSs were cultured on Matrigel or gelatine to better simulate the extracellular environment. Results: The NPs without thioaptamer and conveying 0.1 µg/ml αMG caused a significant dissociation of the MCTSs grown in gelatine after 24 h of treatment (p < 0.01). The most significant disaggregation of MCTSs was obtained using NPs carrying 0.5 µg/ml αMG (p < 0.01). A similar dissociating effect was observed when MCTSs were cultured in Matrigel under the same conditions for 48 - 72 h. By contrast, only concentrations over 1.0 µg/ml of free αMG were able to provoke a damage to MCTSs, consisting in a substantial reduction in their size (p < 0.05). Since the MCTS dissociation induced by αMG-loaded NPs occurred only in the presence of Matrigel or gelatine, an impairment of cell contacts to collagen fibres was likely responsible of this effect. Finally, the treatment of MCTSs with αMG-loaded NPs that were conjugated to the CD44 thioaptamer caused a similar decrease in density but a lower expansion of the spheroid, suggesting that a significant number of cells were died or arrested in cycle. Conclusion: Very low concentrations of αMG delivered by lipidic NPs are sufficient to provoke a substantial disaggregation of MCF-7 MCTSs that involves cell-to-collagen contacts. Similarly, the treatment of MCTSs with NPs conjugated to a CD44 thioaptamer leads to MCTS dissociation but through a more damaging action that causes also a reduction in cell number.

Convergent adaptation of cellular machineries in the evolution of large body masses and long life spans.

In evolutionary terms, life on the planet has taken the form of independently living cells for the majority of time. In comparison, the mammalian radiation is a relatively recent event. The common mammalian ancestor was probably small and short-lived. The "recent" acquisition of an extended longevity and large body mass of some species of mammals present on the earth today suggests the possibility that similar cellular mechanisms have been influenced by the forces of natural selection to create a convergent evolution of longevity. Many cellular mechanisms are potentially relevant for extending longevity; in this assay, we review the literature focusing primarily on two cellular features: (1) the capacity for extensive cellular proliferation of differentiated cells, while maintaining genome stability; and (2) the capacity to detect DNA damage. We have observed that longevity and body mass are both positively linked to these cellular mechanisms and then used statistical tools to evaluate their relative importance. Our analysis suggest that the capacity for extensive cellular proliferation while maintaining sufficient genome stability, correlates to species body mass while the capacity to correctly identify the presence of DNA damage seems more an attribute of long-lived species. Finally, our data are in support of the idea that a slower development, allowing for better DNA damage detection and handling, should associate with longer life span.

E-cigarettes induce toxicological effects that can raise the cancer risk.

Electronic cigarettes (e-cigs) are devices designed to deliver nicotine in a vaping solution rather than smoke and without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few restrictions and a lack of regulatory guidelines. E-cigs have also gained popularity among never-smokers and teenagers, becoming an emergent public health issue. Despite the burgeoning worldwide consumption of e-cigs, their safety remains largely unproven and it is unknown whether these devices cause in vivo toxicological effects that could contribute to cancer. Here we demonstrate the co-mutagenic and cancer-initiating effects of e-cig vapour in a rat lung model. We found that e-cigs have a powerful booster effect on phase-I carcinogen-bioactivating enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), and increase oxygen free radical production and DNA oxidation to 8-hydroxy-2'-deoxyguanosine. Furthermore, we found that e-cigs damage DNA not only at chromosomal level in peripheral blood, such as strand breaks in leucocytes and micronuclei formation in reticulocytes, but also at gene level such as point mutations in urine. Our results demonstrate that exposure to e-cigs could endanger human health, particularly among younger more vulnerable consumers.

6-(Methylsulfonyl) hexyl isothiocyanate as potential chemopreventive agent: molecular and cellular profile in leukaemia cell lines.

Numerous laboratory and epidemiological studies show that the risk of developing several types of cancer can be reduced with the employment of natural substances that act with multiple mechanisms. In this context, an important role is played by the isothiocyanates. Recently, 6-(methylsulfonyl)hexyl isothiocyanate (6-MITC), present in the root of Wasabia Japonica, has stimulated the interest of researchers as a chemopreventive agent. In this particular study we have focused on evaluating 6-MITC's in vitro cytotoxic, cytostatic and cytodifferentiating activities, as well as its pro-apoptotic potential. These effects were investigated by way of flow cytometric analysis of Jurkat and HL-60 cells as well as of healthy lymphocytes extracted from the blood of AVIS donors, in order to verify a potential selectivity of action. The results demonstrate that 6-MITC exerts a stronger cytotoxic effect on tumour cells than on healthy cells. The apoptosis induction exerted by 6-MITC on transformed cells is triggered by an extrinsic pathway, as demonstrated by the statistically significant increase in the percentage of cells with activated caspase-8. It was also observed that 6-MITC is able to limit tumour growth by slowing down and blocking the cell cycle of Jurkat and HL-60 cells respectively, in a dose- and time-related manner, while exerting no activity of any kind on the replication of healthy cells. Finally, by measuring the expression levels of CD-14 and CD-15, 6-MITC showed the ability to induce cytodifferentiation of HL-60 cells into macrophage and granulocytic phenotypes.

DNA Damage Detection by 53BP1: Relationship to Species Longevity.

In order to examine potential differences in genomic stability, we have challenged fibroblasts derived from five different mammalian species of variable longevity with the genotoxic agents, etoposide and neocarzinostatin. We report that cells from longer-lived species exhibit more tumor protein p53 binding protein 1 (53BP1) foci for a given degree of DNA damage relative to shorter-lived species. The presence of a greater number of 53BP1 foci was associated with decreased DNA fragmentation and a lower percentage of cells exhibiting micronuclei. These data suggest that cells from longer-lived species have an enhanced DNA damage response. We propose that the number of 53BP1 foci that form in response to damage reflects the intrinsic capacity of cells to detect and respond to DNA harms.

Genetic instability and aging under the scrutiny of comparative biology: a meta-analysis of spontaneous micronuclei frequency.

In gerontology, comparative biology of longevity offers a powerful observation point thus far underexploited. We use this approach to evaluate the role of genetic stability in longevity determination, extrapolating existing data from the literature. Screening eight pre-existing studies, we collected data from 47 mammalian species and analyzed the relationship of spontaneous micronucleated erythrocyte frequency to species maximum longevity and species adult body mass. Since in 26 of these species the spleen removes micronucleated erythrocytes from the peripheral circulation, we conducted further comparative analysis on the remaining 21 species. We demonstrate that spontaneous micronucleated erythrocyte frequency correlates primarily with body mass and not with maximum longevity. We suggest that other data on genetic stability could be collected from published works in different species and analyzed in a similar way to test further the role of genetic stability in aging.

SEM-Evaluation of enamel surfaces after orthodontic debonding: a 6 and 12-month follow-up in vivo study.

The purpose of this in vivo study was to compare the morphology of the enamel surfaces before bracket bonding and 6 and 12 months after debonding. Replicas of thirty-two maxillary second premolars of 16 volunteers were made before bracket bonding (T0), after debonding (T1), 6 months (T2), and 12 months (T3) later. Scanning electron microscope (SEM) images of the labial enamel surfaces were taken at T0, T1, T2, and T3 at increasing magnifications and analyzed according to the enamel damage index EDI. Data evaluation by using Friedman test followed by Wilcoxon signed ranks test with Bonferroni adjustment did not reveal statistically significant differences in the mean EDI at T0, T2, and T3, whereas the mean EDI at T1 was significantly higher than at T0, T2, and T3 (p < 0.05). The debonding procedure tested in this study produces no clinically relevant enamel damage. These alterations are reversible indeed, as a progressive restoration to pretreatment condition is evident after 6 months already and even more after 12 months.

Interproximal enamel reduction: an in vivo study.

The study aimed to investigate the morphology and composition of the interproximal reduced enamel after exposition to saliva and casein phosphopeptide amorphous calcium phosphate with sodium fluoride (CPP-ACPF). Fourteen patients undergoing an orthodontic treatment with 4 premolars extractions participated to the study. Interproximal enamel reduction (IER) was performed on mesial surfaces of 3 extractive premolars for each patient while 1 served as untreated control. Premolars were assigned to 4 groups: No-S group, sound enamel as control; S-Ex group, stripped and immediately extracted enamel; S-Sal group, stripped and exposed to saliva enamel; S-CPP group, stripped enamel treated with CPP-ACPF. Teeth were extracted at different times, depending on the group they were assigned to and sliced into mesial and distal halves. Mesial surfaces were subjected to environmental scanning electron microscopy with energy dispersive X-ray spectrometry (ESEM/EDX) and to scanning electron microscopy (SEM) analysis. ESEM/EDX investigations showed no statistically significant differences in the content of calcium and phosphate between the 4 groups. SEM observations showed no difference in the morphological appearance of stripped enamel after 30 days of exposure to saliva and CPP-ACPF. Saliva and CPP-ACPF effects on stripped enamel in vivo showed no difference after 30 days.

X-linked hypophosphatemic rickets: enamel abnormalities and oral clinical findings.

X-linked hypophosphatemia (XLH) is a genetic disorder related to alterations in bones and teeth formation, due to low levels of phosphate in blood. Oral findings in XLH have been enamel and dentine abnormalities, high pulp horns, large pulp chambers, and some cases of periapical abscesses related to teeth without caries or traumatic injuries. The aim of our study was to assess the presence of enamel alterations, such as microclefts and/or structure defects in patients with XLH and give guidelines of prevention of XLH dental complications. History taking, oral clinical and radiological examination in 10 young patients affected by XLH (average age of 9) and in 6 patients without XLH (average age of 8). Impressions were performed on the vestibular surfaces of teeth in order to obtain replicas. The replicas were analyzed using scanning electron microscope (SEM) and compared to replicas of control group. The images of replicas of XLH patients showed deep microclefts and irregular enamel surface structure compared to replicas of control group. The replica of a patient with spontaneous periapical abscesses showed numerous enamel crater-shaped depressions and deep microcleavages penetrating into the enamel thickness. In absence of caries or fractures, the abscesses pathogenesis may be related to microcleavages of the enamel and dentin, which allow bacterial invasion of the pulp. There could be a relationship between XLH disease and enamel abnormalities.

Soft tissue cell adhesion to titanium abutments after different cleaning procedures: Preliminary results of a randomized clinical trial

OBJECTIVES: A randomized controlled trial was performed to assess soft tissue cell adhesion to implant titanium abutments subjected to different cleaning procedures and test if plasma cleaning can enhance cell adhesion at an early healing time. Study DESIGN: Eighteen patients with osseointegrated and submerged implants were included. Before re-opening, 18 abutments were divided in 3 groups corresponding to different clinical conditions with different cleaning processes: no treatment (G1), laboratory customization and cleaning by steam (G2), cleaning by plasma of Argon (G3). Abutments were removed after 1 week and scanning electron microscopy was used to analyze cell adhesion to the abutment surface quantitatively (percentage of area occupied by cells) and qualitatively (aspect of adhered cells and presence of contaminants). RESULTS: Mean percentages of area occupied by cells were 17.6 ± 22.7%, 16.5 ± 12.9% and 46.3 ± 27.9% for G1, G2 and G3 respectively. Differences were statistically significant between G1 and G3 (p = 0.030), close to significance between G2 and G3 (p = 0.056), and non-significant between G1 and G2 (p = 0.530). The proportion of samples presenting adhered cells was homogeneous among the 3 groups (p-valor = 1.000). In all cases cells presented a flattened aspect; in 2 cases cells were less efficiently adhered and in 1 case cells presented filipodia. Three cases showed contamination with cocobacteria. CONCLUSIONS: Within the limits of the present study, plasma of Argon may enhance cell adhesion to titanium abutments, even at the early stage of soft tissue healing. Further studies with greater samples are necessary to confirm these findings

Fiber-post bond strength in canals obturated with a cross-linked gutta-percha core obturator.

The aim of this in-vitro study was to evaluate the bond strength of fiber posts cemented in a root canal filled using various root-canal obturation techniques. A total of 33 monoradicular samples, treated endodontically, were randomly assigned to three groups according to the root-canal obturation technique: group 1, continuous-wave technique; group 2, plastic-obturator-core technique; and group 3, cross-linked gutta-percha obturator-core technique. Fiber posts were luted in each sample and each was sectioned perpendicular to the post axis. The push-out test was performed using a universal machine and the maximum failure load was recorded in MPa mm(-2) . Several samples were randomly chosen for scanning electron microscopy evaluation. The mean debris and dentinal tubule-opening scores were calculated separately in the coronal and apical portions. Bond strength was significantly higher in group 1 than in groups 2 and 3. Debris scores were significantly higher in the apical portion of groups 2 and 3 than in group 1. Within the limitations of this study it can be affirmed that thermoplasticized alpha gutta-percha seemed to worsen the cleaning of post-space walls and hence reduced fiber-post bond strength.

A SEM and non-contact surface white light profilometry in vivo study of the effect of a crème containing CPP-ACP and fluoride on young etched enamel.

The aim of this in vivo study was to evaluate the short and a longer term effect on enamel of the application of a crème containing 10% CPP-ACP and 900 ppm fluoride, in orthodontically planned, high caries-risk patients. Epoxy resin replicas of upper lateral incisors were obtained from polyvinyl siloxane (PVS) impressions, before and after etching. The right incisors were left untreated in order to control saliva remineralizing potential. The upper left surfaces were coated with a pea-size amount of the crème. Replicas were obtained at 3 weeks and 6 months and analyzed by SEM and non-contact surface white light profilometry. In the treated sample the profilometric roughness parameters at 3 weeks were statistically significantly lower than the control group values (p < 0.05). At 3 weeks SEM images of the enamel surface showed fewer irregularities. After 6 months, differences between test and control groups were not present on SEM images and profilometric values. CPP-ACP and fluoride crème had positive in vivo effects on enamel surfaces. Significant differences in surface roughness existed after a 3-week period of crème use.

Bracket base remnants after orthodontic debonding.

OBJECTIVE: To evaluate whether the debonding procedure leads to restitutio ad integrum of the enamel surface by investigating the presence of enamel within the bracket base remnants after debonding. MATERIALS AND METHODS: Sixty patients who completed orthodontic treatment with fixed appliances were included. A total of 1068 brackets were microphotographed; the brackets presenting some remnants on the base (n = 818) were selected and analyzed with ImageJ software to measure the remnant area. From this population a statistically significant sample (n = 100) was observed under a scanning electron microscope to check for the presence of enamel within the remnants. Energy dispersive x-ray spectrometry was also performed to obtain quantitative data. RESULTS: Statistically significant differences in the remnant percentage between arches were observed for incisor and canine brackets (P < .0001 and P = .022, respectively). From a morphologic analysis of the scanning electron micrographs the bracket bases were categorized in 3 groups: group A, bases presenting a thin enamel coat (83%); group B, bases showing sizable enamel fragments (7%); group C, bases with no morphologic evidence of enamel presence (10%). Calcium presence was noted on all evaluated brackets under energy dispersive x-ray spectrometry. No significant difference was observed in the Ca/Si ratio between group A (16.21%) and group B (18.77%), whereas the Ca/Si ratio in group C (5.40%) was significantly lower than that of the other groups (P < .323 and P = .0001, respectively). CONCLUSION: The objective of an atraumatic debonding is not achieved yet; in some cases the damage could be clinically relevant.

Evaluation of enamel surfaces after bracket debonding: an in-vivo study with scanning electron microscopy.

INTRODUCTION: The purposes of this in-vivo study were to compare the modes of failure of uncoated and adhesive precoated metal brackets by using the adhesive remnant index, and to assess the quality of the enamel surface after cleanup by using the enamel damage index. METHODS: Twelve Victory brackets (group A) and 12 Victory adhesive precoated brackets (group B) (both, 3M Unitek, Monrovia, Calif) were bonded onto the maxillary second premolars of 12 volunteers. The uncoated brackets were bonded with Transbond XT adhesive resin (3M Unitek). Replicas of the teeth were made before bonding (T0), after bracket removal (T1), and after cleanup (T2). Scanning electron microscope images of all labial enamel surfaces were taken at T0, T1, and T2, and these were evaluated according to the adhesive remnant index and the enamel damage index. RESULTS: Evaluation of the adhesive remnant index scores with the chi-square test showed no statistically significant difference between the groups. Evaluation of the enamel damage index grades with the sign test for paired samples showed a statistically significant difference (P <0.01) between T0 and T2. CONCLUSIONS: Uncoated and precoated brackets exhibited similar debonding patterns. Additionally, the debonding method tested in this study did not restore the original enamel surface, although there was no clinically relevant enamel damage.

A systematic method for predetermined scanning electron microscope analysis in dental science.

Scanning electron microscope evaluation could be criticized if the method adopted to correct for bias is not specified in the study design. Observers can draw conclusions from images unconsciously chosen to best support their research hypotheses, impairing the basic research principle of operator's impartiality. In this study, a systematic observation method has been described and verified for repeatability. The number and the observation points on a certain specimen have been predetermined using a scheme along with observation rules previously established in the research protocol. When our instrument is used at an operating magnification between 500x and 1,000x (corresponding to a frame of 250x190 micro and 120x90 micro, respectively), the method allowed 100% repeatable observation frames, with linear frame errors in finding an observation point of 12.5% in length and 16.8% in height. With modifications to accommodate research objective and statistical requirements, the method could be applied to many SEM observation study.