Antineoplastic drug occupational exposure: a new integrated approach to evaluate exposure and early genotoxic and cytotoxic effects by no-invasive Buccal Micronucleus Cytome Assay biomarker.

Health-care personnel handling antineoplastic drugs could be at risk for adverse health effects. We aimed to evaluate genotoxic and cytotoxic effects of antineoplastic drug exposure of personnel preparing and administering such drugs in three Oncology Hospitals in Italy enrolling 42 exposed subjects and 53 controls. Furthermore, we aimed to study the possible influence of XRCC1 and hOGG1 DNA repair genes polymorphisms on genotoxicity induced on buccal cells. We performed workplace and personal monitoring of some drugs and used exposure diary informations to characterize the exposure. Urinary 5-FU metabolite (α-fluoro-ß-alanine) was measured. Buccal Micronucleus Cytome (BMCyt) assay was used to evaluate DNA damage and other cellular anomalies. GEM and 5-FU contamination was found in 68% and 42% of wipe/swab samples respectively. GEM deposition was found on workers' pads while no α-fluoro-ß-alanine was found. BMCyt-assay showed higher genotoxicity and cytotoxicity on nurses administering antineoplastics than on preparators and controls. Among micronucleus (MN) positive (with MN frequency higher than 1.5‰) exposed subjects, the percentage of those carrying XRCC1 mut/het genotype was higher than in MN positive-controls. Using the sensitive BMCyt assay, we demonstrated that handling antineoplastics still represents a potential occupational health risk for workers that should be better trained/informed regarding such risks.

Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications.

With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 µg/mL for 1, 2, 4 h; 5, 10, 20 µg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 µg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to examine in depth the effects induced by MWCNTs-COOH and, in particular, the timing of the MWCNT-COOH-cell interaction.

[In vitro study of genotoxic and oxidative effects induced on human pulmonary cells by exposure to PAHs extracted from airborne particulate matter collected in a coke plant]. / Studio in vitro degli effetti genotossici ed ossidativi indotti su cellule polmonari umane dall'esposizione ad IPA estratti da matrici ambientali di una cokeria.

Genotoxic and oxidative effect of airborne particulate matter collected in a coke plant were evaluated on lung epithelial cells (A549). We aimed to clarify the mechanism of action of complex mixtures of PAHs and to identify biomarkers of effect of lung cancer. Particulate matter was analysed by GC/MS. Genotoxic and oxidative effects induced by the exposure to the extract were evaluated by Fpg comet assay. The cells were exposed for 30 min, 2h and 4h to 0.01%, 0.02% and 0.05% of the extract. We evaluated comet percentage and analysed tail moment values of exposed and unexposed cells treated with Fpg enzyme (TMenz) and untreated (TM) that indicate respectively oxidative and direct DNA damage. We found 0.328 ng/m3 of pyrene, 0.33 ng/m3 of benzo(a)anthracene, 1.073 ng/m3 of benzo(b)fluoranthene, 0.22 ng/m3 of benzo(k)fluoranthene, 0.35 ng/m3 of benzo(a)pyrene, 0.079 ng/m3 of dibenzo(a,h)anthracene and 0.40 ng/m3 of benzo(g,h,i)perylene. A dose-dependent increase, although not significant, of TM and TMenz in the exposed cells in respect to controls was found that indicates a slight increase of both direct and oxidative damage in exposed cells. A slight increase of comet percentage was found at the highest dose. We show the high sensibility of comet assay to measure early DNA damage also at low doses suggesting the use of such test on A549 to evaluate on target organ the effects of complex mixtures of genotoxic substances.

Sister chromatid exchange and oxidative DNA damage in paving workers exposed to PAHs.

Paving workers are exposed during road paving to several polycyclic aromatic hydrocarbons (PAHs) contained in asphalt fumes. In this study early genotoxic and oxidative effects of exposure to bitumen fumes were evaluated in 19 paving workers and 22 controls. Environmental and biological monitoring of exposure was carried out, measuring, on personal air samples from exposed workers collected during three working days, the concentration of 14 PAHs and urinary OH-pyrene at the end of each of the three working days. Genotoxic effect was evaluated analysing sister chromatid exchange (SCE) frequency and direct-oxidative DNA damage by formamido-pyrimidine-glycosylase (Fpg)-modified comet assay on lymphocytes. Tail moment values from Fpg-enzyme treated cells (TMenz) and from untreated cells (TM) were used as parameters of direct and oxidative DNA damage, respectively. For each subject, the TMenz/TM ratio >2.0 was used to indicate the presence of oxidative damage. DNA damage was also evaluated analysing comet percentage. Personal air samples showed low level of total PAHs (2.843 microg m(-3)) with prevalence of 2-3 ring PAHs (2.693 microg m(-3)). Urinary OH-pyrene after work-shift of the three working days was significantly higher than that found at the beginning of the working week. SCE analysis did not show any difference between two groups while an oxidative DNA damage was found in 37% of exposed with respect to the absence in controls. Comet percentage was significantly higher (P = 0.000 ANOVA) in the exposed than in controls. The results demonstrate the high sensitivity of comet assay to assess early oxidative effects induced by exposure to bitumen fumes at low doses and confirm the suitability of urinary OH-pyrene as a biomarker of PAH exposure. In conclusion the study suggests the use of Fpg-modified comet test as a biomarker of early genotoxic effects and that of urinary OH-pyrene as a biomarker of PAH exposure to furnish indications in terms of characterization, prevention and management of risk in occupational exposure to mixtures of potentially carcinogenic substances.

[Study of genotoxic and oxidative effects induced by PAH exposure in paving workers]. / Studio degli effetti genotossici ed ossidativi indotti dall'esposizione ad IPA in lavoratori addetti alla pavimentazione stradale.

Paving workers are exposed during road paving to several PAHs contained in asphalt fumes. We aimed to evaluate early genotoxic and oxidative effects in 19 paving workers and 22 controls. We analysed sister chromatide exchange (SCE) frequency as marker of genotoxicity. Moreover we assessed oxidative DNA damage by Fpg-modified comet assay on lymphocytes calculating tail moment values from fpg-enzyme treated cells (TMenz) and from untreated cells (TM). For each subject the TMenz/TM ratio higher than 2.0 was used to indicate the presence of oxidative damage. We also evaluated DNA damage analysing comet percentage. SCE analysis didn't show any difference between exposed and control groups. We found oxidative DNA damage in 37% of exposed in respect to the absence in controls. Comet percentage was significantly higher in the exposed than in controls. The results demonstrate the high sensitivity of comet assay to assess early oxidative effects induced by exposure to PAH mixtures at low doses and suggest the use of this biomarker in the characterization, prevention and management of risk induced by occupational exposure to mixtures of potentially carcinogenic substances.

Evaluation of oxidative damage and inhibition of DNA repair in an in vitro study of nickel exposure.

It has been suggested that Nickel is involved in oxidative damage and inhibition of DNA repair. We studied the effects of NiSO4 on oxidative stress and DNA repair in Jurkat cells to elucidate its mechanism of action. Cells were treated with H2O2 and ROS generation (by flow cytometry), and oxidative DNA damage (as tail moment by Fpg-enzyme comet test), were evaluated immediately and after 4 and 24 h of DNA damage recovery occurred in presence or absence of NiSO4 (0.017 and 0.17 microM) to clarify possible interactions of Ni with DNA repair processes. Moreover, cells were exposed to the same doses of NiSO4 for 4 and 24 hours to evaluate its direct oxidative effect. The results of the comet test showed high tail moment immediately after oxidative burst with a decreasing after 4 h of DNA recovery, and a slight increase after 24 h of recovery. The decreases were more limited for cells treated with NiSO4 0.17 microM indicating an inhibition of oxidative DNA damage repair by this substance. An induction of ROS was observed after 4 h of incubation with higher dose of NiSO4. Cells treated with H2O2 showed the highest level of ROS after 4 h of recovery in presence of NiSO4 0.17 microM that remained at elevated levels also after 24 h of recovery suggesting a synergistic action of Ni with H2O2 in the reduction of cellular anti-oxidative defence activities.

[Delphi study in the identification of research need in occupational medicine in Italy]. / Studio Delphi per l'identificazione delle priorità di ricerca in medicina del lavoro in Italia.

The aim of the study was to identify the research priorities and strategies in Occupational Safety and Health (OSH) in Italy with a high degree of consensus. Based on the Delphi technique, a two-phase questionnaire was sent to experts at Universities, Local Health Units (ASLs) and Trade Unions. In the first phase, experts were requested to indicate three research topics in the OSH sector. A total of 27 topics for both universities and ASLs and 18 topics for Trade Unions were identified. In the second phase, experts were requested to assign a score from one (low relevance) to five (high relevance) to each topic previously identified. On the basis of a mean score reported for each topic, two ranked lists of priorities, one referred to universities and ASLs the other referred to Trade Unions, were formulated. The highest priority identified by universities and ASLs was occupational carcinogenesis, followed by quality in occupational medicine. Workers' information, education and participation was also identified with a high degree of priority. For Trade Unions, occupational cancers as well as training, information, participation and prevention awareness had the highest priority. Trade Unions also identify small-industries and occupational accidents as topics with high priority for research development. This study allowed a high degree of consensus to be reached regarding the research priorities in the OSH sector in Italy. Differences in the topics identified, or regarding the mean score of topics commonly identified, were related to the origin of the expert recruited to this study (University, ASLs or Trade Unions) and, for universities and ASLs, to the geographical area. In the authors' opinion, the full transfer of existing scientific data to occupational health practice and the harmonization of the priorities identified by this investigation are crucial if the research needs in the OSH sector in Italy are to be met.

Research priorities in occupational health in Italy.

OBJECTIVE: To find a broad consensus on research priorities and strategies in the field of occupational health and safety in Italy. METHODS: A two phase questionnaire survey was based on the Delphi technique previously described in other reports. 310 Occupational safety and health specialists (from universities and local health units) were given an open questionnaire (to identify three priority research areas). The data obtained from respondents (175, 56.4%) were then used to draw up a list of 27 priority topics grouped together into five macrosectors. Each of these was given a score ranging from 1 (of little importance) to 5 (extremely important). With the mean scores obtained from a total of 203 respondents (65.4%), it was possible to place the 27 topics in rank order according to a scale of priorities. RESULTS: Among the macrosectors, first place was given to the question of methodological approach to research in this field, and for individual topics, occupational carcinogenesis and quality in occupational medicine were ranked first and second, respectively. The question of exposure to low doses of environmental pollutants and multiple exposures ranked third among the priorities; the development of adequate and effective approaches and methods for worker education and participation in prevention was also perceived as being an important issue (fourth place). CONCLUSIONS: This study (the first of its kind in Italy) enabled us to achieve an adequate degree of consensus on research priorities related to the protection of occupational health and safety. Disparities in the mean scores of some of the issues identified overall as being research priorities, seem to be linked both to geographical area and to whether respondents worked in local health units or universities. This finding requires debate and further analysis.

In vivo modulation of ETS genes induced by electromagnetic fields.

We have previously shown that electromagnetic field (EMF) exposure induces ETS1 oncogene overexpression in different cell lines. In order to investigate in vivo EMF effects, BALB/c mice were exposed at different times to 50 MHz radiation, modulated (80%) at 16 Hz. The exposed and control animals were sacrificed and the spleen excised for rt-pcr and western blot analysis. We observed an increase in ETS1 mRNA and protein expression, but a decrease in ETS2 protein levels. Preliminary results from this experimental model show in vivo evidence of the effect of EMF on ETS oncogene expression.

Cytostatic drugs and health risks for exposed personnel: search for new biomarkers.

The use of antiblastic drugs has opened up new perspectives in improvement of therapy and life quality for cancer patients. The widespread clinical application of cytostatic drugs implies risks for exposed hospital personnel, due to genotoxic and toxic-reproductive effects. Biological monitoring is fundamental to identify individuals at risk but is limited by the long latency of chronic effects, absence of unique cellular targets and low sensitivity of available laboratory tests. The objective of this study was to investigate toxic mechanisms by a molecular biology approach, searching for biomarkers potentially useful in monitoring programs. The proposed experimental model consisted of cell line exposure to cyclophosphamide, an alkylating agent of wide clinical use. Cellular response has been investigated focusing on potential targets at RNA level, through reverse transcription polymerase chain reaction (RT-PCR) and differential display analysis. We studied the expression of several genes involved in differentiation, apoptosis and chemoresistance: ets1, bax, bcl-2, bag-1, bcl-X, mdr1 and mrp. Specific patterns of mRNA modulations were observed. Differential display analysis revealed candidate genes induced or repressed following exposure: their characterization is in progress. Besides improving the understanding of toxic mechanisms, identification of modulated molecular targets opens up new perspectives in exposure risk assessment, biomonitoring and preventive strategies at occupational level.

Ets1 oncogene induction by ELF-modulated 50 MHz radiofrequency electromagnetic field.

We have analyzed gene expression in hemopoietic and testicular cell types after their exposure to 50 MHz radiofrequency (RF) non-ionizing radiation modulated (80%) with a 16 Hz frequency. The exposure system generates a 0.2 microT magnetic field parallel to the ground and a 60 V/m electric field orthogonal to the earth's magnetic field. Exposure conditions were selected so as to interfere with the calcium ion flow. Under these electromagnetic field (EMF) conditions, we observed an overexpression of the ets1 mRNA in Jurkat T-lymphoblastoid and Leydig TM3 cell lines. This effect was observed only in the presence of the 16 Hz modulation, corresponding to the resonance frequency for calcium ion with a DC magnetic field of 45.7 microT. We have also identified a putative candidate gene repressed after EMF exposure. The experimental model described in this paper may contribute to the understanding of the biological mechanisms involved in EMF effects.

pH-sensitive imaging by low-frequency EPR: a model study for biological applications.

The use of pH-sensitive nitroxides, in conjunction with low-frequency EPR, offers a unique opportunity for non-invasive assessment of pH values (in the range 0 to 14) in living animals. In the present study, we have investigated the potential use of pH-sensitive nitroxide free radicals in conjunction with EPR imaging techniques at low and very low frequencies (280 MHz-2.1 GHz). In particular, we have measured the hyperfine splitting (hfs) of a pH-sensitive probe at three different EPR frequencies: 280 MHz, 1.1 GHz and 2.1 GHz. We have also developed EPR imaging experiments with phantoms simulating in vivo conditions, using pH-sensitive probes at 280 MHz (spatial-spatial) and 1.1 GHz (spectral-spatial). Finally, we discuss the actual sensitivity/resolution limits of the EPR imaging techniques at low frequencies. Practical applications of this method in the biomedical field are suggested for the continuous and non-invasive localization of pH in vivo.

Simultaneous 280 MHz EPR imaging of rat organs during nitroxide free radical clearance.

A radio frequency (RF) (280 MHz) electron paramagnetic resonance (EPR) spectroscopy and imaging apparatus has been used to localize a pyrrolidine nitroxide free radical in the rat abdomen and thorax. The nitroxide 2,2.5.5,-tetramethylpyrrolidine-1-oxyl-3- carboxylic acid (PCA) had a whole body monoexponential decay with half-life of 13.3 +/- 0.7 (n = 4), 19.4 +/- 0.2 (n = 3), and 23 +/- 2 (n = 6) min for 1, 2, and 3 mmol/kg PCA, respectively. Up to seven one-dimensional longitudinal projections were collected on six rats in the presence of a 8 mT/m field gradient. With an injection dose of 3 mmol/kg, PCA half-lives were 19 +/- 1, 17 +/- 2, and 22 +/- 2 min (n = 6) in the lower abdomen, in the liver, and in the thorax, respectively. Thorax half-life was significantly longer than liver half-life. Sequential two-dimensional images of PCA distribution in a plane longitudinal to the rat body were obtained from eight spectra in the presence of a gradient of 12 mT/m (acquisition time 5 min; spatial resolution 8 mm). After 7 min, the nitroxide was detectable in the left side of the thorax area, but it was mostly localized in the liver. PCA was more uniformly distributed in the image collected after 17 min.

In vivo electron paramagnetic resonance spectroscopy-imaging in experimental oncology: the hope and the reality.

PURPOSE: Low frequency (280 MHz) electron paramagnetic resonance imaging is a new magnetic resonance technique, still being developed, that can map the in vivo spatial distribution of paramagnetic species such as nitroxide free radicals. The reduction rate of these molecules is affected by oxygen concentration. This paper gives some examples of the use of electron paramagnetic resonance imaging methodology in whole rats in the framework of its possible use in experimental oncology. METHODS AND MATERIALS: The 280 MHz apparatus based on a cylindrical 16 pole magnet was developed and designed specifically for 50-200 g laboratory animals. It generates the main field and the three field gradients required for three-dimensional (3-D) projections. A pyrrolidine nitroxyl (2,2,5,5,-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid) was injected intravenously in rats to provide an electron paramagnetic resonance signal for in vivo measurements. Electron paramagnetic resonance X-band spectrometer was used to monitor pyrrolidine nitroxyl decay in an external blood circuit during normoxia and moderate hypoxia (15% O2). RESULTS AND CONCLUSION: One-dimensional (1-D) transversal and longitudinal mapping of this nitroxide free radical distribution in rat whole body was obtained 7-9 min after injection. In circulating blood, nitroxide half-life decreased significantly during hypoxia. The present sensitivity (10(-4)-10(-5) M), spatial resolution (3-10 mm) and collection time (3-5 min) could be drastically improved by narrow linewidth paramagnetic probes and pulsed techniques.

Oxygen-dependent reduction of a nitroxide free radical by electron paramagnetic resonance monitoring of circulating rat blood.

The effects of fraction of inspired oxygen (FiO2) on the reduction of a nitroxide free radical were studied by X-band electron paramagnetic resonance (EPR) monitoring of circulating rat blood. The decay half-life of the metabolism/elimination phase increased significantly by 24 +/- 8% during hyperoxia and decreased significantly by 16 +/- 4% during hypoxia.

Enzymatic synthesis of S-aminoethyl-L-cysteine from pantetheine.

The recently characterized compound S-aminoethylcysteine ketimine can be synthesized from purified S-aminoethylcysteine by enzymatic systems (transaminases or L-amino acid oxidase) present in mammalian tissues. S-Aminoethylcysteine, which could be considered as the natural precursor of the ketimine, is produced from L-serine and cysteamine by the action of the enzyme cystathionine-beta-synthase. We demonstrate in this paper that pantetheine, a normal cellular component, is an efficient cysteamine donor for the synthesis of S-aminoethylcysteine and of S-aminoethylcysteine ketimine in the place of free cysteamine, and we describe the enzymatic system, composed of partially purified enzymes, for the in vitro synthesis of S-aminoethylcysteine ketimine from pantetheine. This seems to indicate a new biological role for pantetheine.