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The purpose of this study of the Italian Society of Reference Values (SIVR) was to provide the reference values for metals in the urine of the Italian general population. Thirteen sampling centres situated in the north, centre and south of Italy took part in this project. Each sampling centre selected 20 adult subjects. The sample was made up of 120 male and 140 female non-smokers or smokers who had abandoned the habit at least 5 years previously, aged between 18 and 60 years. Urine samples were obtained from the same subjects in June and again in November of the same year. During collection of these two samples, we administered an ad hoc questionnaire designed to assess factors that might influence exposure and the results of the study such as personal characteristics, occupational or extra-occupational activities, dietary habits, the presence of dental fillings containing amalgam, the frequency and mode of exposure to passive smoking and exhaust gases of motor vehicles (traffic). Therefore, the urinary levels of thirteen trace elements (Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V) were determined by Inductively Coupled Plasma Mass Spectrometry. The concentrations obtained (95 th percentile) are in μg/L: 0.034, 0.900, 2.24, 0.600, 24.0, 0.013, 1.53, 4.44, 2.64, 0.022, 0.095, 0.759 and 0.855 for Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V, respectively. Further studies with a larger number of subjects are needed in order to continue the biomonitoring of the Italian general population and to monitor the modified levels over time., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder, characterized by severe organ complication. Sickle bone disease (SBD) affects a large part of the SCD patient population, and its pathogenesis has been only partially investigated. Here, we studied bone homeostasis in a humanized mouse model for SCD. Under normoxia, SCD mice display bone loss and bone impairment, with increased osteoclast and reduced osteoblast activity. Hypoxia/reperfusion (H/R) stress, mimicking acute vaso-occlusive crises (VOCs), increased bone turnover, osteoclast activity (RankL), and osteoclast recruitment (Rank) with upregulation of IL-6 as proresorptive cytokine. This was associated with further suppression of osteogenic lineage (Runx2, Sparc). To interfere with the development of SBD, zoledronic acid (Zol), a potent inhibitor of osteoclast activity/osteoclastogenesis and promoter of osteogenic lineage, was used in H/R-exposed mice. Zol markedly inhibited osteoclast activity and recruitment, promoting osteogenic lineage. The recurrent H/R stress further worsened bone structure, increased bone turnover, depressed osteoblastogenesis (Runx2, Sparc), and increased both osteoclast activity (RankL, Cathepsin k) and osteoclast recruitment (Rank) in SCD mice compared with either normoxic or single-H/R-episode SCD mice. Zol used before recurrent VOCs prevented bone impairment and promoted osteogenic lineage. Our findings support the view that SBD is related to osteoblast impairment, and increased osteoclast activity resulted from local hypoxia, oxidative stress, and the release of proresorptive cytokine such as IL-6. Zol might act on both the osteoclast and osteoblast compartments as multimodal therapy to prevent SBD., (© 2015 by The American Society of Hematology.)

N,N-dimethylacetamide (DMA) is used in the textile and plastics industry as a solvent alternative to more toxic N,N-dimethylformamide. Here we studied toxicokinetics of two major urinary metabolites of DMA, namely, S-(acetamidomethyl)mercapturic acid (AMMA) and N-methylacetamide (NMA). Urine samples were collected from workers exposed to DMA in a factory manufacturing acrylic fibers. AMMA and NMA were determined by HPLC/MS and GC/MS, respectively. The working scheme in the factory consisted of periods of three consecutive working shifts alternated regularly with two days off work. In the first stage of the study, NMA and AMMA were determined in urine samples collected before, in the middle, and at the end of one working shift. In the second stage, urine was collected five times during three consecutive days after a two-day rest: before and at the end of the first and second working shifts and before the third shift. It was found that the end-of-shift NMA levels were several folds higher than the pre-shift levels of the same day and dropped significantly until the next shift. On the other hand, there were no significant differences in AMMA levels before and at the end of the same shift but a continuous rise during the three-day working period was observed. Median values of NMA concentrations at the end of working shifts were between 10.1 and 17.3 mg/g creatinine, median AMMA concentrations in the second or third day of the working period varied between 12.4 and 38.1 mg/g creatinine. The approximate half-lives of NMA and AMMA (means) in the exposed workers were about 9 and 29 h, respectively. Thus, while NMA in the end-of-shift urine samples remains a preferential biomarker of DMA exposure during that shift, AMMA determined at the end of a work-week reflects cumulative exposure over the last few days. Further studies are needed to determine AMMA concentrations corresponding to the threshold limit value of DMA., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

The comet assay has been widely used to quantify DNA damage in isolated lymphocytes from subjects exposed to several environmental or occupational substances, especially for estimation of oxidative damage in the DNA, which is well-known to be induced by tobacco smoke. Passive smoking or environmental tobacco smoke (ETS) has been included among those substances that cause cancer with sufficient evidence in humans. In this study, we analyzed, by the alkaline version of comet assay, the lymphocyte DNA damage of white-collar active smokers and non- and ex-smokers exposed to ETS at the workplace. We investigated basal DNA damage, DNA oxidation by formamidopyrimidine glycosylase (Fpg), the repair capacity H2O2-induced DNA damage by kinetics studies and lymphocyte GSH levels, the major intracellular defense against exogenous oxidative stress imposed by cigarette smoking. Our results indicated high basal DNA damage with clear significant correlations with urinary nicotine and cotinine, number of cigarettes/day, and an inverse significant correlation with GSH cellular content in active smokers. Significant Fpg-sensitive sites were found in smokers (> 85%), considerably high but not significant in passive non- and ex-smokers (> 51% and 37%, respectively). The DNA repair capacity had seriously decreased in non-smokers > smokers > ex-smokers, while the same damage was repaired in a short time in never smokers.

Alpha-bisabolol is a compound present in some essential oils, widely distributed in several plants, including camomile. Two different methods for analysing an essential oil, such as alpha-bisabolol in human blood are reported: the first uses micro-liquid chromatography-electrospray ionisation-mass spectrometry (muHPLC-ESI-MS), whereas the second is based on "head space" injection coupled to gas chromatography-mass spectrometry (HS-GC-MS). For LC-ESI-MS, human blood samples, spiked with alpha-bisabolol, were extracted with hexane and evaporated to dryness under air stream. The residue was then reconstituted with methanol and injected into a C18 column, connected to an ion trap mass spectrometer equipped with an ESI source. Spectra were recorded in the positive ion, selected ion monitoring mode. The detection limit of alpha-bisabolol in blood was 0.125 micromol/l. The preparation of samples for the analysis in HS-GC-MS was limited to blood dilution with water (0.5 ml blood + 1 ml water). Head space vials were heated at 125 degrees C for 1 h before automatic injection. The HS-GC-MS detection limit (0.13 micromol/l) was similar to that achieved with the muHPLC-ESI-MS method. Successful tests were performed to verify if alpha-bisabolol could be directly measured by the HS-GC-MS method in different biological samples (blood, urine, faeces, homogenate tissues) from rats treated with the camomile essential oil.

Exposure to 1,3-butadiene (BD), a probable carcinogen to humans, was investigated in two groups of subjects working in a petrochemical plant where BD is produced and used to prepare polymers: 42 occupationally exposed workers and 43 internal non-occupationally exposed controls. BD personal exposure was very low but significantly different in the two groups (median airborne BD 1.5 and 0.4 microg/m(3) in exposed and controls, respectively). Similarly, BD in blood and urine, but not in exhaled air, was higher in the exposed workers than in controls (blood BD 3.7 ng/l versus <1.8 ng/l, urinary BD 2.4 ng/l versus <1.0 ng/l). These three biomarkers correlated significantly with personal exposure ( 0.283 < or = Pearson's r < or = 0.383) and between them (0.780 < or = r < or = 0.896). Excretion of urinary mercapturic acids N-acetyl-S-(3,4-hydroxybutyl)-l-cysteine (MI), N-acetyl-S-(1-hydroxymethyl-2-propenyl)-l-cysteine and N-acetyl-S-(2-hydroxy-3-butenyl)-l-cysteine (MII), chromosomal aberrations (CA), and sister chromatid exchanges (SCE) in peripheral blood lymphocytes were not influenced by occupational exposure. Our results show that unmetabolised BD in biological fluids, and particularly urinary BD, represents the biomarker of choice for assessing occupational exposure to low airborne concentrations of BD.

This paper reviews several procedures for determination of mercapturic acids in urine. Special attention was paid to methods useful in relation to human exposure to industrial pollutants, without any description for less sensitive methods used in animal research. Gas chromatographic and liquid chromatographic procedures were considered together with the little information available about thin layer chromatography and immunochemical techniques. After a description of the main industrial pollutants which lead to synthesis of their specific mercapturic acids, the methods for analysing these products are synthetically reported. The comparison among difficulties in sample preparation, complexity of instrumentation and their cost/benefit ratio are discussed.

A method for the determination of benzene, toluene, ethylbenzene and xylene in blood and urine of people not occupationally exposed to solvents is described. The headspace technique combined with gas chromatography with a mass spectrometer detector is used. The sensitivity of recent mass spectrometers is good enough to furnish reliable results also in biological samples collected from the general population. No treatment for concentrating solvents present in the blood or urine is necessary. The main features of the method are easy preparation of biological samples, small volumes (7 ml), good repeatability and linearity in the range of interest. The limits of detection in blood were 16, 43, 22 and 52 ng/l for benzene, toluene, ethylbenzene and m-xylene respectively. Slightly greater sensitivity was found for urine samples. The results obtained in biological samples from 25 woodworkers not occupationally exposed to BTEX (15 non-smokers and 10 smokers) are comparable to those obtained by other investigators., (Copyright 2002 Elsevier Science B.V.)

Lead and lead compounds play a significant role in modern industry; a wide variety of population is at risk of occupational exposure and lead is suspected to be a human carcinogen. The biochemical and molecular mechanisms of lead toxicity are poorly understood, but emerging data suggest that some of the effects of lead may be due to its interference with calcium in the activation of protein kinase C (PKC) and/or through production of reactive oxygen species (ROS). Many of these results are conducted in vitro on cell lines or ex vivo on human lymphocytes treated in vitro. We, therefore, performed a study on the induction of DNA damage, using the alkaline comet assay, in lymphocytes of battery plant workers. To elucidate in vivo the mechanism(s) responsible for this effect, we determined ROS production, and glutathione (GSH) levels in living cells using the fluorescent probe (2',7'-dichlorofluorescein and monochlorobimane, respectively). Subcellular fractions were obtained from sonicated lymphocytes; cytosolic and membrane expression of PKC isoforms (alpha, and zeta) was evaluated after electrophoresis by immunoblot analysis. The results indicate that lead-exposed workers have significantly elevated levels of DNA breaks compared to the unexposed group. A multivariate analysis of variance (ANOVA) shows that the most common confounding factors (smoking, drinking and age) have no synergistic effects with lead-exposure on the comet parameters or on GSH levels and ROS production. The logistic regression analysis distinguishing the exposed and non-exposed indicates that only GSH with tail moment are selected as significant risk factors. There is a significant positive correlation with ROS production and negative correlation with GSH levels. The content of PKC alpha in cytosol and membranes is decreased 40% (indicating a down-regulation of protein), whereas PKC zeta isoform is not modified in an evident manner. Our results suggest that lead-exposure induces an increase of DNA breakage with an alternate cellular redox state and a significant down-regulation of PKC alpha, suggesting that this metal may act as a tumor promoter.

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-microl volume of urine is added to a carbonate-hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70 degrees C for 10 min. The 'dabsylated' product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C18 column (250 x 4.6 mm, 5 microm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5-40.8 mg/m3), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.

The occupational airborne exposure to benzene of 150 workers employed in petrol stations and a refinery plant was assessed using personal sampling pumps. All workers provided blood samples after the end of work and on the following morning before resuming work. Benzene concentrations in the blood of 243 non-occupationally-exposed subjects were also measured. The median occupational benzene exposure for all 150 workers studied was 80 micrograms/m3. Overall median blood benzene of all workers was 251 ng/l at the end of the shift, and 174 ng/l the following morning. The benzene concentrations measured in blood collected the following morning proved to be significantly lower than those measured at the end of the shift. Median blood benzene for the 243 'normal' subjects was 128 ng/l, which was significantly lower than that measured in the workers before a new work shift. The median blood benzene concentration was significantly higher in smokers than in non-smokers, both in the general population (210 ng/l vs. 110 ng/l) and in the exposed workers at the end of the shift (476 ng/l vs. 132 ng/l) and the following morning (360 ng/l vs. 99 ng/l). End-of-shift blood benzene correlated significantly with environmental exposure; this correlation was better in the 83 non-smokers than in the 67 smokers. In non-smokers with the median benzene occupational exposure of 50 micrograms/m3, no difference was found in blood benzene concentration in exposed and non-exposed subjects.

Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer. Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions. The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes. The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant. Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas. Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding). The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation. Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS). The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples. No mutagenic activity was present in S. typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer. HPLC analysis revealed very low concentrations of PAHs. GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate. We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations. PAHs were not involved in indoor pollution. GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes. These substances are potential carcinogen though their mutagen properties have not been clearly determined., (Copyright 1999 Elsevier Science B.V.)

The analysis of benzene in urine of the general population or of exposed workers can be performed with different methods using the 'purge and trap' or 'solid-phase microextraction' techniques in combination with gas chromatographic analysis and photoionisation or mass spectrometric detection. The published results, however, are deeply conflicting. Differences in sample preparation by different research groups and our own preliminary observations prompted us to investigate pre-analytical and analytical factors potentially capable of modifying the urinary benzene quantification results. Benzene concentrations were measured in 20 urine samples in relation to different conditioning conditions (at 24, 40 and 80 degrees C) and at basic or acid pH. Urinary protein concentrations were measured in the same samples. Urine heating at 80 degrees C yields benzene concentrations on average five times higher than at 24 degrees C. On acidification of urine, the benzene released increases up to 28-fold in comparison to that obtained at uncorrected 'physiological' pH. Despite a widely scattered data distribution, a statistically significant linear correlation was found between 'heat-released' and 'acid-labile' benzene values. There was no correlation between total urinary proteins present in 'physiological' concentrations (between 12 and 110 mg/l) and the different kinds of benzene in urine. Our results could perhaps be explained if it is supposed that part of the benzene in urine is absorbed onto sediment, or bound to specific proteins, or derived from parent molecules and is released with pH modification or heat administration. Our observations may also help to explain why the urinary benzene concentrations reported by different investigators vary considerably even when environmental levels are comparable.