Insights into Cadmium-Induced Carcinogenesis through an In Vitro Study Using C3H10T1/2Cl8 Cells: The Multifaceted Role of Mitochondria.

In this paper, we report the metabolic characterization of two foci, F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 µM CdCl2 for 24 h. The elucidation of the cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. The metabolism of the two foci was investigated through Seahorse and enzyme activity assays; mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. The results showed that F1 focus has higher glycolytic and TCA fluxes compared to F3 focus, and a more negative mitochondrial membrane potential, so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 focus showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. Overall, our results showed that cadmium treatment induced lasting metabolic alterations in both foci. Triggered by the loss of the Pasteur effect in F1 focus and by mitochondrial impairment in F3 focus, these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.

Somatization in a primary care service for immigrants.

AIM: To study somatization in a large sample of immigrants attending a first visit to a primary care service. Differences in somatization among four large immigrant groups (Europeans, Asians, South Americans, and Africans) and 16 subgroups based on nationality were assessed. DESIGN: A total of 3105 patients were asked to participate in the study, of whom 3051 completed the 21-item version of the Bradford Somatic Inventory (BSI-21). Patients scoring 14 or higher on the BSI-21 were considered to be somatizers. A multiple logistic regression analysis adjusting for intervening variables tested the relative risk of somatization in and among the groups. RESULTS: Among the 3051 patients who completed the BSI-21, 782 (25.6%) were somatizers. Somatizers were significantly more prevalent among South Americans (30.1%). After adjusting for covariates, Asians and Europeans, but not Africans, showed a significantly lower risk of somatization compared to South Americans. Among national subgroups, somatization occurred more frequently in Peruvians (32.9%). Compared to Peruvians, migrants from Eastern Europe, Morocco, the Philippines, Sri Lanka, and El Salvador demonstrated a significantly lower risk of somatization. CONCLUSIONS: Approximately one-fourth of socially disadvantaged immigrants who accessed primary care services used somatization to express their distress. However, the likelihood of somatization varied widely among the different groups, and was significantly higher in South Americans and in some African groups, and lower in some Asian groups.

Fluorescence of KCl Aqueous Solution: A Possible Spectroscopic Signature of Nucleation.

Ion pairing in water solutions alters both the water hydrogen-bond network and ion solvation, modifying the dynamics and properties of electrolyte water solutions. Here, we report an anomalous intrinsic fluorescence of KCl aqueous solution at room temperature and show that its intensity increases with the salt concentration. From the ab initio density functional theory (DFT) and time-dependent DFT modeling, we propose that the fluorescence emission could originate from the stiffening of the hydrogen bond network in the hydration shell of solvated ion-pairs that suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway. Because computations suggest that the fluorophores are the local ion-water structures present in the prenucleation phase, this band could be the signature of the incoming salt precipitation.

Anomalous Intrinsic Fluorescence of HCl and NaOH Aqueous Solutions.

The unique properties of liquid water mainly arise from its hydrogen bond network. The geometry and dynamics of this network play a key role in shaping the characteristics of soft matter, from simple solutions to biosystems. Here we report an anomalous intrinsic fluorescence of HCl and NaOH aqueous solutions at room temperature that shows important differences in the excitation and emission bands between the two solutes. From ab initio time-dependent density functional theory modeling we propose that fluorescence emission could originate from hydrated ion species contained in transient cavities of the bulk solvent. These cavities, which are characterized by a stiff surface, could provide an environment that, upon trapping the excited state, suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway.

Two-dimensional versus three-dimensional morphometry of monogenoidean sclerites.

A new method of three-dimensional (3-D) analysis of sclerotised structures of monogenoids was performed by processing z-series images using 3D-Doctor. Z-series were obtained from Gomori's trichrome-stained specimens of marine and freshwater monogenoids under laser scanning confocal fluorescence microscopy. Measurements obtained from 3-D images were then compared with those from 2-D images taken from both flattened and unflattened specimens. Data comparison demonstrated that 3-D morphometry allowed avoidance of over-estimation due to deformation and the reduction of errors associated with different spatial orientations. Moreover, study of 3-D images permitted observation of morphological details that are not detectable in 2-D representations.

Novel nontoxic mitochondrial probe for confocal fluorescence microscopy.

We propose a 2,5-Bis[1-(4-N-methylpyridinium)ethen-2-yl)]-N-methylpyrrole ditriflate (PEPEP) as a novel nontoxic, nonpotentiometric mitochondrial probe for confocal fluorescence microscopy. PEPEP is a representative chromophore of a large family of heterocyclic fluorescent dyes that show fluorescence emission in aqueous media and great DNA affinity. We check its cytotoxicity and intracellular localization in mammalian and yeast cell cultures. We demonstrate that PEPEP is a very efficient dye for fluorescence confocal microscopy and a valuable alternative to the most frequently used mitochondrial stains.

Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy.

A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy.

Mitochondria in tumor cells studied by laser scanning confocal microscopy.

We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine. We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology. In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery. Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells. We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein. In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be seen in this cell line. Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology. Our results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity.

Ethidium bromide as a marker of mtDNA replication in living cells.

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.

Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene films: chemical characterization and evaluation of the protein adsorption.

This work deals with the optimization of argon plasma-induced graft-polymerization of polyethylene glycol acrylate (PEGA) on polypropylene (PP) films in order to obtain surfaces with a reduced protein adsorption for possible biomedical applications. To this end, we examined the protein adsorption on the treated and untreated surfaces. The graft-polymerization process consisted of four steps: (a) plasma pre-activation of the PP substrates; (b) immersion in a PEGA solution; (c) argon plasma-induced graft-polymerization; (d) washing and drying of the samples. The efficiency of these processes was evaluated in terms of the amount of grafted polymer, coverage uniformity and substrates wettability. The process was monitored by contact angle measurements, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray Photoelectron Spectroscopy (XPS) and atomic force microscopy (AFM) analyses. The stability of the obtained thin films was evaluated in water and in Phosphate Buffer Saline (PBS) at 37 degrees C. The adsorption of fibrinogen and green fluorescent protein (GFP)--taken as model proteins--on the differently prepared surfaces was evaluated through a fluorescence approach using laser scanning confocal microscopy with photon counting detection. After plasma treatments of short duration, the protein adsorption decreases by about 60-70% with respect to that of the untreated film, while long plasma exposure resulted in a higher protein adsorption, due to damaging of the grafted polymer.

Ethidium bromide as a vital probe of mitochondrial DNA in carcinoma cells.

The interaction of ethidium bromide (EB) with mitochondria in human breast and lung carcinoma cells was investigated under living conditions, employing a laser scanning confocal fluorescence microscopy (LSCFM) with a photon counting detection to reduce drastically the laser power excitation and the fluorescent probe concentration. In sensitive and multidrug-resistant MCF-7 cell lines, which are important model systems for the study of mitochondria in tumour cells, two distinct populations of mitochondria were observed, each characterised by a different EB fluorescence, membrane potential, cellular localisation and morphology. By image analysis, these peripheral mitochondria showed a peculiar morphology, consisting of punctuate organelles (0.8mum in size) organised in rosette-like assemblies. Unexpectedly, an intense EB fluorescence was observed in these mitochondria, indicating a high accessibility to EB of their mtDNA, which is likely to be in an active replicative or transcriptional state. These results might, therefore, suggest an active biogenesis and metabolism of the peripheral mitochondria that could be a consequence of the increased energetic needs of the cells, after their tumour transformation. Indeed, the pool of peripheral mitochondria, as well as their peculiar morphology and spatial organisation, was found to be a characteristic feature of all the carcinoma cells examined here, but not of their non-transformed parental MCF10A cells.