Cytocompatibility and cellular internalization mechanisms of SiC/SiO2 nanowires.

First evidence of in vitro cytocompatibility of SiC/SiO2 core-shell nanowires is reported. Different internalization mechanisms by adenocarcinomic alveolar basal epithelial cells, monocytic cell line derived from an acute monocytic leukemia, breast cancer cells, and normal human dermal fibroblasts are shown. The internalization occurs mainly for macropinocytosis and sporadically by direct penetration in all cell models considered, whereas it occurred for phagocytosis only in monocytic leukemia cells. The cytocompatibility of the nanowires is proved by the analysis of cell proliferation, cell cycle progression, and oxidative stress on the cells treated with NWs as compared to controls. Reactive oxygen species generation was detected as an early event that then quickly run out with a rapid decrease only in adenocarcinomic alveolar basal epithelial and human dermal fibroblasts cells. In all the cell lines, the intracellular presence of NWs induce the same molecular events but to a different extent: peroxidation of membrane lipids and oxidation of proteins. The NWs do not elicit either midterm (72 h) or long-term (10 days) cytotoxic activity leading to irreversible cellular damages or death. Our results are important in view of a possible use of SiC/SiO2 core-shell structures acting as biomolecule-delivery vectors or intracellular electrodes.

[Biological monitoring and exposure to silica: application of new dose and effect biomarkers]. / Monitoraggio biologico ed esposizione a silice: applicazione di nuovi indicatori di dose e di effetto.

Si-CAE was measured in 16 composite marble industry workers furthermore, a spirometry was performed and 8oxoGua, 8oxoGuo 8oxodGuo, SP-A, SP-D, CC16 and HO-1 were dosed. A lower spirometric values (FEV1 and FVC) were observed among workers compared with controls and the following markers were increased: Si-CAE, 8oxoGuo and HO-1 expression. This study shows that exposure to silica can increase the levels of Si-CAE, which can be used to estimate the dose to the target. Finally, nonspecific spirometric abnormalities and an increase in biomarkers of effect were observed.

A comparison between the effects of over-expression of miRNA-16 and miRNA-34a on cell cycle progression of mesothelioma cell lines and on their cisplatin sensitivity.

The prognosis of patients affected by malignant pleural mesothelioma (MPM) is presently poor and no therapeutic strategies have improved their survival yet. Introduction of miRNA mimics to restore their reduced or absent functionality in cancer cells is considered an important opportunity and a combination of miR's might be even more effective. In the present study, miR-16 and miR-34a were transfected, singularly and in combination, in MPM cell lines H2052 and H28, and their effects on cell proliferation and sensitivity to cisplatin are reported. Interestingly, the overexpression of both miRs, alone or combined, slows down the cell cycle progression, modulates the p53 and HMGB1 expression and increases the sensitivity of cells to cisplatin, producing a marked impairment of cell proliferation and strengthening the apoptotic effect of the drug. However, the co-overexpression of the two miRs results more effective only in the regulation of the cell cycle, but does not enhance the sensitivity of MPM cells to cisplatin. Consequently, although the potential of miR-16 and miR-34a is confirmed, we must conclude that their combination does not improve the response of MPM to chemotherapy.

A cytotoxicity study of silicon oxycarbide nanowires as cell scaffold for biomedical applications.

GOAL: Nanowires are promising biomaterials in multiple clinical applications. The goal of this study was to investigate the cytotoxicity of carbon-doped silica nanowires (SiOxCy NWs) on a fibroblastic cell line in vitro. MATERIALS AND METHODS: SiOxCy NWs were grown on Si substrates by CVD process. Murine L929 fibroblasts were cultured in complete DMEM and indirect and direct cytotoxicity tests were performed in agreement with ISO 19003-5, by quantitating cell viability at MTT and chemiluminescent assay. Cell cultures were investigated at Scanning Electron Microscope (SEM) and immunocytochemistry to observe their morphology and investigate cell-NWs interactions. Furthermore, hemocompatibility with Platelet-rich Plasma was assayed at SEM and by ELISA assay. RESULTS: SiOxCy NWs proved biocompatible and did not impair cell proliferation at contact assays. L929 were able to attach on NWs and proliferate. Most interestingly, L929 reorganised the NW scaffold by displacing the nanostructure and creating tunnels within the NW network. NWs moreover did not impair platelet activation and behaved similarly to flat SiO2. CONCLUSIONS: Our data show that SiOxCy NWs did not release cytotoxic species and acted as a viable and adaptable scaffold for fibroblastic cells, thus representing a promising platform for implantable devices.

Analysis of oxidative stress in SK-N-MC neurons exposed to styrene-7,8-oxide.

Styrene-7,8-oxide (SO) is the main metabolite of styrene, a neurotoxic volatile organic compound used industrially. Here we report the novel observation that several markers of oxidative stress were affected in SK-N-MC cells exposed for 16 h to concentrations of SO that induce apoptotic cell death. The production of Thiobarbituric Acid Reactive Substances (TBARS), rose from 69.1 +/- 15.7 nmol/g protein (control) to 119.3 +/- 39.2 and 102.0 +/- 17.3 nmol/g protein after exposure to 0.3 and 1 mM SO, respectively. Carbonyl group levels were significantly enhanced by SO at both concentrations. The lower dose also decreased sulphydryl groups. SO caused a marked oxidative DNA damage, as shown by a fivefold increase in 8-hydroxy-2(')-deoxyguanosine (8-OHdG). In addition, SO exposure resulted in alterations of scavenging abilities, given the reduction of both glutathione (GSH) and glutathione-S-transferase (GST) activity. Induction of expression of the oxidative stress response gene heme-oxygenase-1 (HO-1) and an increased HO-1 activity were also observed. These data provide compelling evidence that oxidative stress significantly contributes to SO toxicity in neuronal cells.

[Neuroendocrine and renal effects of inorganic lead]. / Effetti neuroendocrini e renali del piombo inorganico.

In this study 371 workers occupationally exposed to inorganic Pb (range of blood lead concentrations, PbB: 100-800 microg/l) were examined in order to assess neuroendocrine and renal effects, with regard to exposure levels and ALAD polymorphism. Plasma prolactin, urinary excretion of plasmaproteins and renal tissue constituents were measured. None of such markers differed significantly between workers stratified according to PbB levels, except for heat-stable isoenzyme NAG-B: its very low prevalence of values above the upper reference limit increased significantly with increasing PbB. No significant differences were found in indicators by ALAD genotype. Our findings did not provide evidence of any renal and neuroendocrine effects in workers exposed to the current lead levels.

[Chronobiological evaluation of effects biomarkers and sampling]. / Importanza della valutazione cronobiologica degli indicatori di effetto per una corretta strategia di campionamento.

Occupational exposure to oxidants is often associated with an increase in the levels of oxidative DNA damage in urine. Besides 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), other products of position-8 guanine oxidation have been identified in urine, including 8-hydroxy-guanine (8-oxo-G) and 8-hydroxyguanosine (8-oxy-Guo). The aim of the present study was the characterization of these effect biomarkers in terms of inter- and intra-individuals varaibility, as well as in terms of their excretion profile during a 24 h-period. Urine samples were collected from 11 volunteers (6 samples/day). Urine concentrations of 8-oxo-G, 8-oxo-Guo, and 8-oxo-dG were determined by liquid chromatography-tandem mass spectrometry. The inter-individual variability, expressed as variation coefficient, was 85-150% for 8-oxo-G, 20-45% for 8-oxo-Guo, and 30-45% for 8-oxo-dG. The statistical anaysis for repeated measurements showed that none of the biomarkers was affected by significant variation during the day (one-way ANOVA, p < 0.05), thus excluding the existence of a circadian rhythm. We conclude that the sampling time is not critical for the assessment of oxidative DNA damage in urine.

[ALAD polymorphism and indicators of dose and effects of occupational exposure to inorganic lead]. / Polimorfismo ALAD ed indicatori di dose e di effetto dell'esposizione professionale a piombo inorganico.

The delta-aminolevulinate dehydrase (ALAD) genetic polymorphism was investigated along with biomarkers of lead exposure and effect on 333 male workers, occupationally exposed to lead, with blood lead levels (PbB) higher than 100 microg/l. ALAD genotype frequencies were distributed as expected among Caucasians. Workers bearing at least one ALAD-2 allele showed PbB values slightly higher than those from ALAD-1-1 subjects, who also exhibited higher urinary delta-aminolevulinic acid (ALAU) and blood zinc protoporphyrin (ZPP) levels. The plasmatic lead (PbP)/PbB ratio was similar in both groups. Exposure and effect biomarkers were significantly each other correlated among ALAD-1-1 subjects only, who showed also a significant inverse relationship between ALAD activity and ZPP values. Results confirm previous studies, supporting the hypothesis that ALAD polymorphism may interfere with toxico-kinetic and toxico-dynamic parameters of occupational exposure to Pb.

[Report on the activities carried out in the research project of the Ministry of Instruction, University, and Research entitled "Environmental and occupational exposure to inorganic lead: assessment of toxic effects of current doses and related preventive measures"]. / Presentazione delle attività svolte nell'ambito del progetto di ricerca MIUR "Esposizione ambientale ed occupazionale a piombo inorganico: studio degli effetti tossici conseguenti alle dosi correnti e delle relative misure preventive".

It is here presented the project sustained by the Ministry of Public Instruction, University and Research "Environmental and Occupational Exposure to Inorganic Lead: assessment of human health effects due to current doses and preventive measures" ruled out between 2001 and 2003. The aim of the study was to investigate about the toxic effects related to current occupational exposures to inorganic lead (particularly those effects concerning blood pressure, carcinogenic risk, nervous and immunological systems), to identify hypersusceptibility conditions, particularly the ALAD genetic polymorphism and to evaluate the role of traditional biomarker and the possibility of introducing new ones. In the present article the procedures followed during the project lasting and the contribution of each Unit are described. The results of the research, presented in detail in the current issue, do confirm the inadequacy of the biological exposure index nowadays ruled by Lex 25/2002.

Exhaled breath analysis in suspected cases of non-small-cell lung cancer: a cross-sectional study.

Lung cancer is one of the most frequently diagnosed cancers worldwide and is still the leading cause of cancer-related deaths. There is a considerable interest in finding diagnostic methods in the disease's earliest stages. A complementary approach to imaging techniques could be provided by exhaled breath gas phase and exhaled breath condensate (EBC) analysis. The aim of this study was to quantify various biomarkers in the exhaled breath gas phase and EBC in suspected cases of non-small-cell lung cancer (NSCLC). The study involved 138 subjects with suspected lung cancer, 71 of whom had a subsequent diagnosis of NSCLC. The diagnostic power of a combination of hydrogen peroxide (H2O2)-EBC, and exhaled pentane, 2-methyl pentane, hexane, ethyl benzene, heptanal, trans-2-nonenal in distinguishing NSCLC and non-NSCLC subjects was poor-to-fair (area under the curve (AUC) = 0.68), similar to that of smoking history alone (expressed as pack-years, AUC = 0.70); a further improvement was observed when smoking history was combined with exhaled compounds (AUC = 0.80). The diagnostic power was increased in those patients with little or no past smoke exposure (AUC = 0.92) or where past smoke exposure was up to 30 pack-years (AUC = 0.85). Exhaled substances had a good accuracy in discriminating suspected cancerous cases only in those subjects with a modest smoking history (≤ 30 pack-years), but the inclusion of other exhaled biomarkers may increase the overall accuracy, regardless of tobacco smoke.

Porphyrin conjugated SiC/SiOx nanowires for X-ray-excited photodynamic therapy.

The development of innovative nanosystems opens new perspectives for multidisciplinary applications at the frontier between materials science and nanomedicine. Here we present a novel hybrid nanosystem based on cytocompatible inorganic SiC/SiOx core/shell nanowires conjugated via click-chemistry procedures with an organic photosensitizer, a tetracarboxyphenyl porphyrin derivative. We show that this nanosystem is an efficient source of singlet oxygen for cell oxidative stress when irradiated with 6 MV X-Rays at low doses (0.4-2 Gy). The in-vitro clonogenic survival assay on lung adenocarcinoma cells shows that 12 days after irradiation at a dose of 2 Gy, the cell population is reduced by about 75% with respect to control cells. These results demonstrate that our approach is very efficient to enhance radiation therapy effects for cancer treatments.

Urinary excretion of brush-border antigen and plasma proteins in early stages of diabetic nephropathy.

In 109 patients with insulin-dependent diabetes mellitus (IDDM), we measured the urinary excretion of albumin, the low molecular weight proteins (LMWP) retinol-binding protein (RBP) and beta 2-microglobulin (beta 2m), and brush-border antigens (BBA) revealed by monoclonal antibodies. All such markers of kidney damage and/or dysfunction were higher in diabetic patients than in 44 controls. Increased urinary levels of BBA (p = 0.0001) were associated with higher values of albumin (p = 0.0002), RBP (p = 0.0005) and, to a lesser extent, of beta 2m (p = 0.1), different combinations of values above the reference limits being observed. Some 30 and 40% of patients with and without microalbuminuria, respectively, also exhibited signs of tubulopathy. Although under certain circumstances tubular defects may give rise to small increases in albuminuria, the most likely explanation for our findings is the coexistence of glomerular and tubular damage in some patients with IDDM. Neither the prognostic value nor the pathophysiological meaning of tubular damage and/or dysfunction can be assessed by the present study, owing to its cross-sectional design. Tubular markers thus deserve further studies to clarify whether in diabetic patients they indicate a more severe or diffuse kidney impairment.

Multiple interferences on catecholamine metabolism by tetrahydroisoquinolines (TIQs).

TIQs are thought to be formed by condensation between dopamine and certain metabolites of ethanol, organic solvents and anesthetic gases. Described here are experiments aimed at evaluating TIQs interference with catecholamine synthesis. Rat adrenal pheochromocytoma (PC12) cell lysates were exposed to benzyl-TIQ and phenyl-TIQ. The activities of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) were measured by HPLC-based methods following exposure to variable concentrations of TIQs. The effects of TIQs on DBH activity were also assessed in human serum. Dixon plot analyses revealed that TIQs act on TH as competitive inhibitors with different affinity. Ki for benzyl- and phenyl-TIQ were 5 and 3 microM respectively. DBH activity in serum exposed to benzyl- and phenyl-TIQ ranging from 0.2 to 20 microM rose respectively by 12.5% to 58% for benzyl- and by 7.8% to 26% for phenyl-TIQ. Such TIQs interferences with catecholamine metabolism seem to account for dopamine (DA) depletion observed in parallel in vitro experiments on PC12 cells. The dose-dependent inhibition of TH and the increased activity of DBH together with the relatively low effective doses of TIQs suggest this mechanism as a possible explanation of the selective toxicity of styrene and other solvents to dopaminergic systems observed in rabbits following experimental exposure and suspected to occur in occupationally-exposed workers.

Dopamine (DA) metabolism in PC12 cells exposed to manganese (Mn) at different oxidation states.

The present study was aimed at assessing the role of Mn valency state in Mn-induced changes in DA metabolism by PC12 cells. Mn(ll)Cl2, Mn(lll)Acetate, and Mn(IV)O2 were used for these experiments. PC12 cells were incubated for 3, 24 and 72 hours to Mn nominal concentrations ranging from 10-8 to 10(-4) M in 24-well plates containing 2 x 10(5) cells/well. Supernatants and cellular materials were then separated and immediately processed for the analysis of dopamine (DA), and its metabolite 3,4-di-hydroxy-phenylacetic acid (DOPAC). Lactate dehydrogenase (LDH) activity and MTT cleavage were measured as indices of cell death. In parallel experiments, Mn-containing medium (10(-5) M) was removed and cells incubated for further periods with Mn-free medium to evaluate the reversibility of observed changes. At the end of the experimental periods, none of Mn-exposed cultures showed appreciable reduction in cell viability as compared to their respective controls. After exposure to Mn(II) and Mn(III), irreversible and dose-dependent decreases in the medium but not in intra-cellular DA were apparent. Indeed, 10(-4) M Mn(II) caused the disappearance of DA and DOPAC from the medium. The same effect was caused by 10(-5) M Mn(III), the dose-effect relationship being shifted towards lower dose levels. Mn(IV) induced a parallel and dose-dependent decrease of DA and DOPAC concentrations in both intra- and extra-cellular compartments. Such an effect was reversible after removal of Mn from the medium. Multiple interferences on DA metabolism are caused by Mn. Mn(II) and Mn(III) seem to block DA secretion without affecting DA turnover rate. Mn(IV) seems to cause DA depletion and aspecific (secondary) changes in secretion rates. Further studies are necessary to understand the mechanisms underlying the differential effects of various Mn compounds on DA metabolism.

An in vitro model for the assessment of manganese neurotoxicity.

PC12 (undifferentiated and differentiated) and C6 cells have been used to investigate kinetics, morphological and functional endpoints following exposure to MnCl(2) and manganic transferrin (Mn-Tf). [Mn](i) in undifferentiated (non-differentiated cells) exposed to both free (MnCl(2)) and bound Mn (Mn-Tf), was three- to fivefold lower as compared to differentiated (differentiated) PC12 cells and higher by one order of magnitude as compared to glial C6 cells. Exposure to both MnCl(2) and Mn-Tf was followed by time- and dose-dependent morphological changes characteristic of apoptosis, which was never observed in Mn-exposed C6 glial cells. Results from cell viability assays were consistent with apoptotic response rates quantified by cell count. Threshold concentrations for undifferentiated and differentiated PC12 cells were 10(-6) and 10(-5)m, respectively. Thus, despite their greater ability to accumulate Mn, differentiated PC12 cells are less sensitive to Mn-induced apoptosis. This model might be relevant to neuronal degeneration induced by Mn occurring in the developing brain and possibly in clinical manganism. Such critical doses at the cellular level seem to be consistent with Mn levels (5x10(-6)m) recorded in the basal ganglia of monkeys chronically exposed to Mn and developing clinical signs of manganism.

Serum prolactin in subjects occupationally exposed to manganese.

To evaluate whether or not occupational exposure to manganese (Mn) affects basal levels of serum prolactin (PRL), a cross-sectional study was carried out in 31 occupationally-exposed workers, aged 39.2 years (DS 7.9) exposed to manganese (Mn) dusts for 14.5 years (range: 5 to 29 years) in a ferroalloy producing plant. Thirty-four industrial workers not exposed to neurotoxic chemicals and of comparable age composed the control group. Airborne Mn concentrations in dusts of the furnace area ranged 210 to 980 micrograms/m3, which is below the current American Conference of Governmental Industrial Hygienists (ACGIH)-recommended threshold limit value-time weighted average (TLV-TWA) of 1 mg/m3. Manganese concentrations in blood Mn (MnB) and in urine (MnU) were significantly higher in Mn-exposed workers as compared to control workers. The Mn-exposed workers showed significantly higher serum prolactin (PRL) levels with the geometric mean (GM) being 9.77 ng/ml with a geometric standard deviation (GSD) of 1.69 as compared to controls (GM 4.65 ng/ml, GSD 1.78, p < 0.001). Serum PRL was negatively related to age and positively correlated with both MnB and MnU. Dose-effect relationships were still significant in partial correlation analysis after control for age. The prevalence of abnormally high PRL values was consistent with a dose-response relationship. The observed increase in serum PRL among Mn-exposed workers suggests an impairment of tonic inhibition by tubero-infundibular dopaminergic neurons. The correlation between PRL and both MnB and MnU in samples collected at least 48 h from the last exposure suggests that such indices provide an estimation of the target dose.

Reference values for early markers of renal damage.

In addition to the difficulties related to the definition of a reference population and hence of reference limits, special methodological problems have to be dealt with when early markers of renal damage are applied to monitor groups at risk. The urinary excretion of both high and low molecular weight proteins, as measured by sensitive immunoassays, is routinely applied in health surveillance programmes aimed at preventing the occurrence of adverse renal effects resulting from exposure to nephrotoxic chemicals, among which are several metals. Owing to the large intra-individual variability experimentally demonstrated for all indicators of renal impairment, carefully standardized sampling conditions must be adopted. At the individual level, repeated measurements should be performed before a diagnosis of renal disease or dysfunction is established. In fact, several physiological conditions may account for transient changes in renal function, leading to measured levels greatly exceeding reference limits.

Does occupational cobalt exposure determine early renal changes?

A cohort of workers occupationally exposed to cobalt (Co) dusts was examined to assess possible subclinical renal effects attributable to Co. Cross-sectional investigations involved 26 workers with a mean age of 34.2 (S.D., 8.3), chronically-exposed (median, 3.5 years; range, 0.9-11) to Co dusts in hard-metal manufacturing factories. Thirty-five healthy control workers, with a mean age of 32.4 (S.D., 4.6) were also examined. Individual interviews were used to exclude subjects with renal or systemic diseases, intake of nephrotoxic drugs, and exposure to known nephrotoxins. Exposure levels, assessed by ambient and biological monitoring, showed an estimated exposure approaching the ACGIH-recommended TLV of 50 micrograms/m3. Immunochemical methods were used to measure urinary albumin, retinol-binding protein (RBP), beta 2-microglobulin (beta 2m), and tubular brush-border antigens. The prevalence of abnormal values for early markers of renal dysfunction was similar in Co-exposed workers and in controls. However, within the reference interval, the cumulated frequency distribution for beta 2m was shifted towards higher values in the exposed group. No relationship was detected between renal markers and either intensity or duration of exposure. In spite of a limited number of observations, these findings suggest that the kidney is not a target organ during occupational exposure to Co.

Development and validation of new screening tests for nephrotoxic effects.

Within the framework of an European Commission-funded project, groups of industrial workers exposed to heavy metals (cadmium, mercury and lead) or solvents were studied together with corresponding control groups. Eighty-one measurements were carried out on urine and serum samples and the scientific results together with individual questionnaire information were entered into a central database. Data obtained was assessed centrally and individually in subsidiary studies. The measurable contributions were assessed either singly or in combination, of smoking, gender, metal exposure and site, to nephrotoxicity. The potential value of each test as an indicator of nephrotoxicity was then assessed on the basis of sensitivity and specificity. A number of new tests including prostaglandins and for extracellular matrix components were investigated as well as established tests for renal damage and dysfunction. The data obtained from this comprehensive study emphasises the value of noninvasive biomarkers for the early detection of nephrotoxicity due to environmental toxins. The urinary profile varied with the type of environmental/occupational toxin. By careful selection of a small panel of markers they can be used to indicate the presence of renal damage, the principal region affected, and to monitor the progress of disease and damage. Biomarkers were also used to confirm and tentatively establish safe exposure levels to nephrotoxins.

Urinary excretion of tubular brush-border antigens among lead exposed workers.

The brush-border of the human renal proximal tubule is extremely sensitive to toxic, ischaemic and inflammatory insults. A monoclonal antibody to a brush-border antigen (BB-50) had been shown to identify increased urinary excretion of BB-50 (UBB-50) among workers exposed to heavy metals and hydrocarbons as well as patients on cisplatin and patients with early diabetic nephropathy. This study describes the use of this antibody to quantify UBB-50 among lead exposed workers. The study population consisted of 154 workers from a factory manufacturing lead stabilisers. Of these 91 workers had less than 6 months of exposure and formed the control group. The remaining 63 workers with a median exposure of 3 years formed the exposed group. Several blood lead (PbB) indices were used as exposure indices. These include the most recent PbB (PbBrec), a time-integrated blood lead index (PbBint), an absolute change in recent PbB (PbB delta), a relative change in PbB (PbB delta%), as well as the number of times the PbB measurements were above 40, 50 and 60 micrograms/100 ml (PbB40, PbB50, PbB60 respectively). Urinary levels of beta-2-microglobulin (U beta 2m), alpha-1-microglobulin (U alpha 1m), retinol binding protein (URBP), albumin (UAlb), activity of total N-acetyl-D-beta-glucosaminidase (NAG-T) and heat stable NAG isoenzyme (NAG-B) were measured along with the serum beta 2m (S beta 2m). Through stepwise analysis, UBB-50 was best correlated with PbBint, PbB40 and PbB delta% (r2, 0.271). Of these, PbBint and PbB40 had about twice the contribution to the variation in UBB-50.(ABSTRACT TRUNCATED AT 250 WORDS)