Antonio Colombi

The speciation of the chemical forms of arsenic in the biological monitoring of exposure to inorganic arsenic

Total As content may be determined in blood and urine by means of an AAS method that involves reduction of As to its volatile hydride and ashing at 600 degrees C with MgO and Mg (NO3)2. Separation of inorganic As (InAs), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMAA) by ion-exchange chromatography, followed by direct AAS analysis, allows the determination of each As species in the urine. In a reference population of 148 subjects with only normal environmental exposure to As, total As concentration in the urine averages 17.2 +/- 11.1 micrograms/l. Urinary As consists of 10% each of InAs, MMAA and DMAA, the remaining 70% consisting of other forms of organic As. Blood As concentration averages 5.1 +/- 6.9 micrograms/l and correlates significantly with the urinary concentration of InAs and the sum of its metabolites (InAs + MMAA + DMAA). Inorganic arsenic undergoes methylation in the organism. After ingestion of high quantities of As2O3, the time course of excretion of its metabolites indicates that As methylation occurs by a saturable mechanism. In workers exposed to As2O3, InAs, MMAA and DMAA are the only chemical forms of As excreted in the urine that are relevant to a study of occupational exposure. Blood As concentration is proportional to exposure and correlates only with urinary DMAA excretion; DMAA seems to be the most appropriate single indicator of exposure. At high levels of exposure (total As excretion above 200 micrograms/l), As accumulates in the organism and DMAA excretion reflects its accumulation. At low levels of exposure (total As excretion below 50 micrograms/l) a short-term accumulation does not occur and the best biological indicator of exposure is InAs excretion. Seafood ingestion brings about a marked increase in urinary excretion of total As that lasts for 24-48 h and is not accompanied by any increase in InAs, MMAA or DMAA excretion. Organic As from seafood does not mix with the pool of inorganic As in the organism and may be separately detected in urine. In the biological monitoring of human exposure to As, particularly in the case of high urinary values, the speciation of the chemical forms of As in urine is necessary in order to establish with certainty the source, industrial or alimentary, of exposure.

Monitoring low benzene exposure : comparative evaluation of urinary biomarkers, influence of cigarette smoking and genetic polymorphisms

Benzene is a human carcinogen and an ubiquitous environmental pollutant. Identification of specific and sensitive biological markers is critical for the definition of exposure to low benzene level and the evaluation of the health risk posed by this exposure. This investigation compared urinary trans,trans-muconic acid (t,t-MA), S-phenylmercapturic acid, and benzene (U-benzene) as biomarkers to assess benzene exposure and evaluated the influence of smoking and the genetic polymorphisms CYP2E1 (RsaI and DraI) and NADPH quinone oxidoreductase-1 on these indices. Gas station attendants, urban policemen, bus drivers, and two groups of controls were studied (415 subjects). Median benzene exposure was 61, 22, 21, 9 and 6 μg/m3, respectively, with higher levels in workers than in controls. U-benzene, but not t,t-MA and S-phenylmercapturic acid, showed an exposure-related increase. All the biomarkers were strongly influenced by cigarette smoking, with values up to 8-fold higher in smokers compared with nonsmokers. Significant correlations of the biomarkers with each other and with urinary cotinine were found. A possible influence of genetic polymorphism of CYP2E1 (RsaI and/or DraI) on t,t-MA and U-benzene in subjects with a variant allele was found. Multiple linear regression analysis correlated the urinary markers with exposure, smoking status, and CYP2E1 (RsaI; R2 up to 0.55 for U-benzene). In conclusion, in the range of investigated benzene levels (<478 μg/m3 or <0.15 ppm), smoking may be regarded as the major source of benzene intake; among the study indices, U-benzene is the marker of choice for biomonitoring low-level occupational and environmental benzene exposure.

Environment And Genetics in Lung cancer Etiology (EAGLE) study: an integrative population-based case-control study of lung cancer.

BackgroundLung cancer is the leading cause of cancer mortality worldwide. Tobacco smoking is its primary cause, and yet the precise molecular alterations induced by smoking in lung tissue that lead to lung cancer and impact survival have remained obscure. A new framework of research is needed to address the challenges offered by this complex disease.Methods/DesignWe designed a large population-based case-control study that combines a traditional molecular epidemiology design with a more integrative approach to investigate the dynamic process that begins with smoking initiation, proceeds through dependency/smoking persistence, continues with lung cancer development and ends with progression to disseminated disease or response to therapy and survival. The study allows the integration of data from multiple sources in the same subjects (risk factors, germline variation, genomic alterations in tumors, and clinical endpoints) to tackle the disease etiology from different angles. Before beginning the study, we conducted a phone survey and pilot investigations to identify the best approach to ensure an acceptable participation in the study from cases and controls. Between 2002 and 2005, we enrolled 2101 incident primary lung cancer cases and 2120 population controls, with 86.6% and 72.4% participation rate, respectively, from a catchment area including 216 municipalities in the Lombardy region of Italy. Lung cancer cases were enrolled in 13 hospitals and population controls were randomly sampled from the area to match the cases by age, gender and residence. Detailed epidemiological information and biospecimens were collected from each participant, and clinical data and tissue specimens from the cases. Collection of follow-up data on treatment and survival is ongoing.DiscussionEAGLE is a new population-based case-control study that explores the full spectrum of lung cancer etiology, from smoking addiction to lung cancer outcome, through examination of epidemiological, molecular, and clinical data. We have provided a detailed description of the study design, field activities, management, and opportunities for research following this integrative approach, which allows a sharper and more comprehensive vision of the complex nature of this disease. The study is poised to accelerate the emergence of new preventive and therapeutic strategies with potentially enormous impact on public health.

Headspace solid-phase microextraction for the determination of benzene, toluene, ethylbenzene and xylenes in urine

A method for the determination of benzene, toluene, ethylbenzene and xylenes (BTEX) in urine of people exposed to these airborne pollutants present in the living environment, has been described. Solid-phase microextraction has been used for sampling BTEX from the headspace of urine and gas chromatography-mass spectrometry has been applied for the selective analysis of chemicals. The method has the following features: small volume of urine (2 ml) needed, linearity in the range of interest (from the limit of detection up to 5000 ng/l) with coefficient of correlation > or =0.998, limit of detection in the range 12-34 ng/l, good repeatability (coefficient of variation 2-7%), high specificity. The stability of the urine sample during storage (-20 degrees C) was evaluated: BTEX remained stable for up to 2 months. The assay has been successfully applied to the biological monitoring of two subjects environmentally exposed to airborne BTEX in an urban area.

Biological and environmental monitoring of exposure to airborne benzene and other aromatic hydrocarbons in Milan traffic wardens

Environmental and biological monitoring of airborne aromatic hydrocarbons has been performed in 20 policemen working as traffic wardens exposed to motor vehicle exhausts and in 19 peers employed as clerks. Airborne benzene, toluene, ethylbenzene and xylene concentrations, measured during the workshift, resulted in significantly higher outdoor than indoor concentrations (benzene and related aromatic hydrocarbons mean values, respectively of 53 and 350 micrograms/m3 vs. 29 and 180 micrograms/m3). Blood benzene, toluene, ethylbenzene and xylene concentrations did not differ significantly between indoor and outdoor workers; no differences were found between values obtained at the beginning (07:30 h) and the end of shift (00:30) in either group. Blood hydrocarbon concentrations seem to reflect airborne pollution, whilst the blood benzene concentration determined after the workshift poorly reflects airborne benzene morning peaks. Endshift blood benzene mean concentration in smokers (462 ng/l, n = 9) differs significantly from non-smokers (292 ng/l, n = 39).

Toxicological and immune findings in workers exposed to Pentachlorophenol (PCP)

Pentachlorophenol (PCP) is a pesticide used worldwide in industrial and domestic applications. Data available on the effects of technical-grade PCP on the immune system are insufficient and equivocal; some data indicate inhibitory effects, whereas others suggest stimulating effects. This study was performed to evaluate toxicological and immune findings in 32 subjects who had prolonged exposure to PCP in a wood factory and in 37 controls. PCP concentrations were determined in plasma and urine of all subjects. Lymphocyte subsets of CD3-, CD4-, and CD8-positive cells were evaluated, and the proliferative response of peripheral blood mononuclear cells (PBM) to mitogens was assessed. The results suggested the absence of major laboratory and clinical signs of PCP-dependent immune deficiency. A weak effect of long-term exposure to PCP on the functional immune response could not be ruled out because of the finding of a decreased response to 5% PHA in the high-exposure group. A weak effect against hepatocyte membrane was evidenced by the finding of raised serum concentration of glycocholic, taurodeoxycholic, and glycochenodeoxycholic acids in subjects directly exposed to PCP for more than 10 y.

Inactivation of fecal bacteria in sewage sludge by alkaline treatment

The efficacy of aqueous alkaline solutions to purify wastewater sludges destined for agricultural land application was evaluated. Some fecal bacteria were tested as sanitary-quality indices, these included fecal streptococci, which were found to be more resistant than coliforms. Hygienization within 10 days was obtained with ammonium hydrate at a dose that brought the pH of the sludge to about 10. Such a treatment was effective above 10°C. The total-bacteria number dropped only slightly, so that the sludge retained its potential biological activity. The efficacy of the treatment with NH4OH was better than that observed with KOH.

Neurobehavioural effects of pesticides with special focus on organophosphorus compounds: which is the real size of the problem?

The risk of neurobehavioural impairment as a consequence of a prolonged, low dose exposure to neurotoxic pesticides is not clearly demonstrated despite numerous publications addressing the topic. We reviewed the 24 papers published on human neurobehavioural effects of organophosphorus and/or carbamates pesticides up to May 1st 2008. Variables evaluated were compound/s addressed, number of subjects, approach to measure or estimate exposure, characteristics of control groups and presence of confounders, methodological approach, and type of alteration, taking into account cognitive, sensory-motor, psychological, and psychomotor measures. A total of 6 papers considered the whole spectrum of functions, the studies yielding positive or uncertain results were 13 (68%) for cognitive function, 11 (69%) for psychomotor function, 11 (65%) for sensory-motor function, and 11 (65%) for psychological function impairment. In 46% of the positive studies a previous severe acute poisoning was reported. Exposure levels were measured only in 5 studies, and very often there were problems in the selection of controls, and firm conclusions on the risk of neurobehavioural effects cannot be reached yet. The main limits of the available data are: limited number of studies and compounds addressed, significant differences in the approach among studies, poor concordance of the results of different studies, and difficulties in controlling confounding factors. Nevertheless, there are sufficient data to conclude that neurobehavioural impairment might be the consequence of an acute poisoning, and possibly the consequence of relatively high and prolonged exposures.

High-performance liquid chromatography of methotrexate for environmental monitoring of surface contamination in hospital departments and assessment of occupational exposure

In the frame of applicative research in occupational hygiene of hospital workplaces, we investigate hospital indoor contamination as a consequence of the use of antineoplastic drugs (ANDs), with the purpose of assessing exposure of medical and nursing personnel to potentially harmful doses of ANDs, and ultimately of yielding advice on safe operating procedures for manipulation of ANDs in hospitals and in house-care of cancer patients. Among the large number of currently employed ANDs, methotrexate (MTX) has been selected as a tracer of surface contamination, on the basis of its wide use in therapy, its ease of measurement and of its chemical properties relevant to persistence and transport in the indoor environment. MTX is a polyelectrolyte, with a high water, but lower organic solvent solubility, a negligible vapour pressure and a high chemical robustness to environmental stress, thus allowing to measure surface-to-surface carryover (e.g. from spillage or glove fingerprint) and indoor contamination due to aerosol transport (e.g. from syringe manipulation procedures). Monitoring of MTX in environmental samples such as swab washings of surfaces and objects requires an analytical method with characteristics of sensitivity, reproducibility, precision, analytical speed, ease of automation and robustness. We have therefore developed an analytical procedure which employs simple short-column RP-HPLC with UV detection, automated sample injection and a close analogue internal standard for improved precision and solid-phase extraction (SPE) for sample concentration. Our method has proven suitable for detecting traces of MTX on a wide variety of surfaces and objects, with a limit of quantification in the range of 50 microg/dm3 for direct injection of unconcentrated washings, corresponding to the possible detection of surface contamination as low as 1 microg/m3 and a limit of detection in the range of 10 ng/m2 for samples as large as 100 dm3 concentrated by SPE. We present preliminary results from a recent hospital case-study, assessing the contamination level of furniture and equipment in drug preparation areas. Spillage fractions as high as 5% of the employed mass (70-260 mg/day) are measured on the polythene-backed paper disposable hood cover sheet; traces of MTX in the microgram range can also be measured on floor surfaces, furniture and handles, even at a distance from the preparation hoods.

Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans, trans-Muconic acid (1,3-butadiene-1, 4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C18 column (5 x 0.46 cm I.D., 3 microns particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50-500 microgram/l range; the quantification limit was 6 micrograms/l; day-to-day precision, at 300 micrograms/l, was C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean +/- S.D. = 77 +/- 54 micrograms/l, n = 82) were statistically different from those of smokers (169 +/- 85 micrograms/l, n = 30) (P < 0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.