Luigi Perbellini

Reference values for exhaled nitric oxide (reveno) study.

BackgroundDespite the widespread use of fractional exhaled nitric oxide (FENO) as a biomarker of airways inflammation, there are no published papers describing normal FENO values in a large group of healthy adults.ObjectiveThe aim of this study was to establish adult FENO reference values according to the international guidelines.MethodsFENO was measured in 204 healthy, non-smoking adults with normal spirometry values using the on-line single-breath technique, and the results were analysed chemiluminescently.ResultsThe main result of the study was the significant difference in FENO values between men and women, thus indicating that gender-based reference FENO values are necessary. The FENO levels obtained at expiratory flows of 50 ml/s ranged from 2.6 to 28.8 ppb in men, and from 1.6 to 21.5 ppb in women.ConclusionWe propose reference FENO values for healthy adult men and women that could be used for clinical and research purposes.

Application of high performance liquid chromatography/tandem mass spectrometry in the environmental and biological monitoring of health care personnel occupationally exposed to cyclophosphamide and ifosfamide

Twenty four workers (10 involved in the preparation and 14 in administration) exposed to cyclophosphamide (CP) and ifosfamide (IF) in two Italian hospitals were monitored. The extent of exposure was assessed by the analysis of air samples, wipe samples, pads and gloves. Urinary excretion at the beginning and at the end of the work shift was also measured by liquid-liquid extraction and analysis by high performance liquid chromatography/tandem mass spectrometry. Three out of 24 air samples were positive for CP or IF. In wipe samples, CP concentrations ranging from < 0.001 to 82.4 micrograms/dm2 in Hospital A (32 samples) and from 0.2 to 383.3 micrograms/dm2 in Hospital B (17 samples), were found. IF concentrations varied from < 0.001 to 90.9 micrograms/dm2 in Hospital A and from 0.01 to 141.5 micrograms/dm2 in Hospital B. Pads (from 11 to 13 for each operator) were contaminated with CP and IF especially on arms, legs and chest. The use of a plastic-backed liner on the working tray in the laminar flow hoods was demonstrated to compromise the containment properties of the hood. Urine samples were positive for CP in 50% of the workers (range: 0.1-2.1 micrograms/L), whereas IF was detected in 2 subjects only (range: 0.1-0.8 microgram/L). The results of this investigation demonstrate that vertical laminar airflow hoods, when incorrectly used, might represent a source of contamination and that higher risk may depend on lack of educational programmes and observance of preventive guidelines.

Breath and blood levels of benzene, toluene, cumene and styrene in non-occupational exposure

SummaryBenzene, toluene, cumene and styrene were measured in the breath and blood of two groups of individuals. The first group included individuals belonging to a hospital staff, the second group included chemical workers who were not exposed to the abovementioned chemicals. The chemical workers were examined in plant infirmaries on the morning before the start of the workshift, and the hospital staff in the hospital infirmaries. One environmental air sample was taken in the infirmaries for each individual at the moment of the biological samplings. The environmental concentrations of benzene and styrene were significantly higher in the infirmaries of the chemical plant than in the infirmaries of the hospital. On the other hand, the environmental concentrations of toluene and cumene were not significantly different in the plant infirmaries and in the hospital infirmaries. In the hospital staff the alveolar concentrations of benzene, toluene and styrene were significantly lower than those in the chemical workers. In the hospital staff the blood concentrations of benzene, toluene and styrene were not significantly different from those in the chemical workers. Only the blood cumene concentration was significantly higher in the chemical workers. In hospital staff, smokers showed alveolar and blood concentrations of benzene and toluene that were significantly higher than those measured in the non smoker hospital staff. With reference to chemical workers, only alveolar benzene concentration was significantly higher in smokers than in non smokers. A significant blood benzene difference was found between the non smoker hospital staff and the non smoker chemical workers. A correlation between alveolar and environmental concentrations was found for benzene, toluene and cumene, but not for styrene. In the two groups of individuals, correlations between blood and alveolar concentrations of the four compounds were also studied.

Reference values for blood benzene in the occupationally unexposed general population.

SummaryBlood benzene was determined by gas chromatography-mass spectrometry in 431 “normal” subjects, subdivided into 155 rural subjects and 276 urban subjects. Blood benzene (mean value 262 ng/l) was significantly lower in rural (200 ng/l) than in urban (296 ng/l) workers, as well as differing significantly between 293 non-smokers and 138 smokers (205 ng/l and 381 ng/l, respectively). Among non-smokers, values were significantly higher (307 ng/l) in 76 chemical workers. In the total study population, in 95% of cases blood benzene was less than 718 ng/l, the 95th percentile being 514 ng/l in non-smokers vs 901 ng/l in smokers and 576 ng/l in rural vs 822 ng/l in urban subjects. Within each population subgroup, the difference between non-smokers and smokers was statistically significant, except among office workers (non-smokers 234 ng/l, smokers 304 ng/l). Blood benzene (y) was directly proportional to the number of cigarettes smoked (x) (y = 201 + 12x; r = 0.44; n = 431), and inversely proportional to the interval between the last cigarette and the time at which the blood sample was taken (z) (log y = 6.167 − 0.0015 z; r = −0.461; n = 135). The blood half-life of benzene was about 8h. The multiple correlation between blood benzene (Cb), number of cigarettes per day (x) and time since the last cigarette (z) is: Cb = 417 + 7.2x − 0.41z (n = 135; R = 0.20; P < 0.00001).

Liquid-liquid extraction procedure for trace determination of cyclophosphamide in human urine by high-performance liquid chromatography tandem mass spectrometry

A sensitive, specific and accurate high performance liquid chromatography/ionspray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine from hospital personnel involved in drug preparation and administration of antineoplastic alkylating agents. This methodology, which includes liquid-liquid extraction with ethylacetate, requires no derivatization procedures, preventing cyclophosphamide (CP) from possible thermal and chemical decomposition reactions. We detected the excretion of this unmetabolized alkylating drug in 50% of all the study participants. The amount of CP ranged from 0.1 ng microL-1 to 1.9 ng microL-1 urine. This methodology was validated by the use of ifosfamide as internal standard. The assay was linear over the range 0 to 3.2 ng microL-1 urine, with a lower limit of quantification of 0.2 microL-1. The limit of detection was assessed at 0.05 ng microL-1 urine. This method is characterized by a coefficient of variation < 10%. Standard calibration curves, obtained on three different days, had correlation coefficients always greater than 0.998. The intra and interday precision were within 11%, and accuracy was in the range 99-103%. The mean extracted recovery assessed at three different concentrations (0.5, 0.8, 3.2 ng microL-1) was always more than 85%. The extraction efficiency of cyclophosphamide from urine samples was also studied at six different pH values (pH 4, 5, 6, 7, 8, 10). The maximum extraction efficiency was obtained when the pH of urine solutions was adjusted to 7.0

The validity of urinary metabolites as indicators of low exposures to toluene.

SummaryExposure to toluene was studied in a group of 14 subjects working in a printing industry, who were exposed to this solvent only. Environmental monitoring was carried out using personal samplers for the whole workshift over three consecutive days. Toluene TWA concentrations ranged from 37 to 229 mg/m3. At the end of the workshift on each day of investigation, urine samples were collected for the determination of hippuric acid and ortho-cresol. Hippuric acid was also determined for urine before the workshift and on the Saturday and Monday mornings after the end of exposure; hippuric acid was also determined in 16 controls over the same five-day period. At the end of the workshift, hippuricuria levels in exposed workers always turned out to be statistically different from pre-workshift levels and those of the controls.The end-of-workshift hippuricuria levels of exposed workers were significantly correlated with the mean daily environmental concentration (TWA): in the three days of comparative study, we found r = 0.63 (P < 0.05) on Day 1, r = 0.90 (P < 0.001) on Day 2, and r = 0.87 (P < 0.001) on Day 3. Orthocresol turned out to be correlated with daily exposure less significantly than hippuric acid: r = 0.49 (n.s.) on Day 1; r = 0.78 (P < 0.001) on Day 2, and r = 0.65 (P < 0.05) on Day 3. Using all available data (41 observations), a very significant correlation (P < 0.001) was found between the TWA and both metabolites (r = 0.80 for hippuric acid; r = 0.68 for o-cresol). The values of the two metabolites in the end-of-workshift urine samples (41 observations) also turned out to be well correlated (r = 0.70; P < 0.001). The authors conclude that hippuric acid is a valid test for evaluating even low exposures to toluene.

Insight into the apoptosis-inducing action of α-bisabolol towards malignant tumor cells: Involvement of lipid rafts and Bid

In a precedent report we showed that alpha-bisabolol, a sesquiterpene present widely in the plant kingdom, exerts a rapid and efficient apoptosis-inducing action selectively towards human and murine malignant glioblastoma cell lines through mitochondrial damage. The present study extends these data demonstrating the apoptosis-inducing action of alpha-bisabolol towards highly malignant human pancreatic carcinoma cell lines without affecting human fibroblast viability. The present study further shows the preferential incorporation of alpha-bisabolol to transformed cells through lipid rafts on plasma membranes and, thereafter, direct interaction between alpha-bisabolol and Bid protein, one of pro-apoptotic Bcl-2 family proteins, analyzed either by Surface Plasmon Resonance method or by intrinsic fluorescence measurement. Notions that lipid rafts are rich in plasma membranes of transformed cells and that Bid, richly present in lipid rafts, is deeply involved in lipid transport make highly credible the hypothesis that the molecular mechanism of alpha-bisabolol action may include its capacity to interact with Bid protein.

Physiologicomathematical model for studying human exposure to organic solvents: kinetics of blood/tissue n-hexane concentrations and of 2,5-hexanedione in urine.

The physiologicomathematical model with eight compartments described allows the simulation of the absorbtion, distribution, biotransformation, excretion of an organic solvent, and the kinetics of its metabolites. The usual compartments of the human organism (vessel rich group, muscle group, and fat group) are integrated with the lungs, the metabolising tissues, and three other compartments dealing with the metabolic kinetics (biotransformation, water, and urinary compartments). The findings obtained by mathematical simulation of exposure to n-hexane were compared with data previously reported. The concentrations of n-hexane in alveolar air and in venous blood described both in experimental and occupational exposures provided a substantial validation for the data obtained by mathematical simulation. The results of the urinary excretion of 2,5-hexanedione given by the model were in good agreement with data already reported. The simulation of an exposure to n-hexane repeated five days a week suggested that the solvent accumulates in the fat tissue. The half life of n-hexane in fat tissue equalled 64 hours. The kinetics of 2,5-hexanedione resulting from the model suggest that occupational exposure results in the presence of large amounts of 2,5-hexanedione in the body for the whole working week.

Involvement of mitochondrial permeability transition pore opening in α‐bisabolol induced apoptosis

α‐Bisabolol is a natural monocyclic sesquiterpene alcohol. It has been used in cosmetics for hundreds of years because of its perceived skin‐healing properties. α‐Bisabolol is known to have anti‐irritant, anti‐inflammatory and antimicrobial properties. In precedent studies, we described how α‐bisabolol exerts a selective pro‐apoptotic action towards transformed cells [Cavalieri E et al. (2004) Biochem Biophys Res Commun315, 589–594] and its uptake is mediated by lipid rafts on the plasma membrane [Darra E et al. (2008) Arch Biochem Biophys476, 113–123]. In this study, we hypothesize that the intracellular target of α‐bisabolol may be the mitochondrial permeability transition pore (mPTP). To evaluate this hypothesis, we used one transformed cell line (human glioma T67) in comparison with a nontransformed one (human fibroblasts). We assessed the effect of a specific mPTP inhibitor (cyclosporine A) on the toxic action of α‐bisabolol. Results show that the α‐bisabolol‐induced decrease in oxygen consumption is abolished by the addition of cyclosporine A in T67 cells, indicating that α‐bisabolol may target mPTP. The central role of mitochondria was also demonstrated by using galactose to force cells to a more aerobic metabolism. In this condition, we observed higher α‐bisabolol toxicity. Furthermore, we studied the effect of α‐bisabolol on isolated rat liver mitochondria. This study expands the notion of the specific action of α‐bisabolol on transformed cells and suggests that it may act by disturbing the structure and function of the mPTP. α‐Bisabolol toxicity is clearly related to its cellular uptake, which is higher in transformed cell lines.

Toluene concentrations in the blood and alveolar air of workers during the workshift and the morning after

Occupational toluene exposure was studied during the workshift and the morning after by the analysis of environmental air, alveolar air, and blood. Environmental toluene exposure was measured by both continuous and instantaneous sampling. Instantaneous environmental toluene concentrations correlated better with alveolar toluene concentrations (r = 0.94; n = 155) than with blood toluene concentrations (r = 0.71; n = 52). Continuous environmental toluene concentrations correlated better with blood toluene concentrations (r = 0.84; n = 65) than with alveolar toluene concentrations (r = 0.52; n = 46). During the workshift and the morning after, blood and alveolar toluene concentrations correlated significantly with each other (r = 0.75; n = 66 and r = 0.67; n = 52). In a group of workers who were exposed to a mean environmental toluene concentration of 146 micromilligrams the concentrations of toluene in the alveolar air and blood the morning after were 3.2 micromilligrams (SD = 1.7) and 27.5 micromilligrams (SD = 12.7) respectively. With regard to the morning after toluene determinations, blood concentrations correlated (r = 0.52; n = 52; p less than 0.001) better than the alveolar concentrations with the corresponding afternoon values (r = 0.36; n = 52; p less than 0.01). The decline of the toluene concentrations from the end of one workshift to the start of the next exposure indicated a mean toluene half life of 3.8 hours in the alveolar air and of 4.5 hours in blood and therefore the 17 hour interval between two consecutive workshifts was insufficient for the complete elimination of absorbed toluene.