Occupational exposure to zoonotic agents among agricultural workers in Lombardy Region, northern Italy.

OBJECTIVES: This study was conducted in Northern Italy with the aim of defining the risk of agricultural workers' contact with biological agents through the determination of serum antibodies against selected zoonotic agents. Immunity against tetanus was also investigated. METHODS: Two groups of agricultural workers consisting of 153 animal breeders (exposed) and 46 non- breeders (controls) were included in the study. In a first group of 103 workers (89 exposed and 14 controls) the serum concentrations of antibodies against Hepatitis E Virus (HEV) were measured, whereas in the second group of 96 workers (64 exposed and 32 controls) the serum concentrations of antibodies against Leptospira spp., Coxiella burnetii, Borrelia burgdorferi, Brucella spp. and Salmonella spp. were addressed. Imunization against tretanus was also studied in this group. RESULTS: Animal breeders showed higher rates of IgG antibodies against Coxiella burnetii (50% vs. 31.2%), and Leptospira spp. (59.4% vs. 43.7%). Results of logistic regression analysis revealed that breeder workers showed a tendency to have higher prevalence of positivity for antibodies to Leptospira spp.and Coxiella burnetii than non-breeders (ORs ~ 3). Only one exposed subject showed antibodies against hepatitis E (none in controls), but when tested with another commercially available kit the percentage of anti HEV IgG positive subjects increased to 22.3% in the exposed, while none of the controls showed positive. None of the subjects showed antibodies against Salmonella spp. and Brucella spp. Italians and other European workers have better protection against tetanus (91%) compared to non-EU workers (81%). CONCLUSIONS: The higher frequency of the presence of serum antibodies to zoonotic agents (e.g. Leptospira spp. and Coxiella burnetii) in animal breeders suggests that they are more exposed to biological agents than workers not involved in animal breeding activities. The risk of contact with HEV deserves further studies because the adoption of different assays can result in significantly different results. The promotion of immunization of agricultural workers might be a priority, in particular for migrants.

Persistent organochlorinated pesticides and mechanisms of their toxicity.

Persistent organic pollutants comprised of organic chemicals like polychlorinated biphenyls, dibenzo-p-dioxins, dibenzofurans and organochlorinated pesticides which have many characteristics in common. Once released in the environment they resist physical, biological, chemical and photochemical breakdown processes and thus persist in the environment. They are subject to long transboundary air pollution transport. They accumulate in the food chain due to their lipophilicity, bioaccumulation and biomagnification properties. Human exposure occurs through inhalation of air, ingestion of food and skin contact. Because most of them bioaccumulate and remain preferentially in fat, their long-term effects are still a matter of public health concern. They are condemned for health adverse effects such as cancer, reproductive defects, neurobehavioral abnormalities, endocrine and immunological toxicity. These effects can be elicited via a number of mechanisms among others include disruption of endocrine system, oxidation stress and epigenetic. However most of the mechanisms are not clear thus a number of studies are ongoing trying to elucidate them. In this review, the underlying possible mechanisms of action and their possible roles in adverse developmental and reproductive processes are discussed and where possible a linkage is made to some existing epidemiological data. Both genomic and nongenomic pathways are used to describe these effects. Understanding of these mechanisms will enable development of strategies to protect the public by reducing these adverse effects. This review is limited to persistent organochlorinated pesticides (OCPs) such as dichlorodiphenyltrichloroethane (DDT) and its metabolites, hexachlorobenzene (HCB), beta-hexachlorocyclohexane (ß-HCH) and endosulfan.

Emerging zoonoses: the "one health approach".

Zoonoses represent a public health risk recently pointed out by the spreading of previously unknown human infectious diseases emerging from animal reservoirs such as severe acute respiratory syndrome and avian influenza caused by H5N1-virus. These outbreaks have shown that animal breeding activities can pose a significant public health risk. Until now, the risk of zoonoses has probably been underestimated, particularly in occupational settings. The emergence or re-emergence of bacterial (Mycobacterium bovis and Brucella spp) or viral (hepatitis E virus) infections shows that zoonoses should be considered as emerging risks in agricultural and animal breeding and should be addressed by specific preventive interventions. Close cooperation and interaction between veterinarians, occupational health physicians and public health operators is necessary, for a worldwide strategy to expand interdisciplinary collaborations and communications in all aspects of health care for humans, animals and the environment. This is what the One Health Approach was intended to be.

Immune changes in animal breeders: a pilot study conducted in northern Italy.

OBJECTIVE: Farming is associated with exposure to a wide variety of risk factors including organic dusts, endotoxins, allergens and other chemicals. The ability of some of these agents to interact with the immune system is demonstrated in the presented study which was undertaken to evaluate the relationship between pig and cow breeding, and the immune system early changes. Particular attention is paid to selected serum cytokines. METHODS: Sixty four animal breeders (36 cattle and 28 pig breeders) were selected as the exposed group, and 32 rural workers not engaged in animal breeding were utilised as the controls. Personal data were collected through a questionnaire, and selected serum parameters measured, including cytokines IL-6, IL-8, IL-10, IFNγ and TNFα, immunoglobulins and proteins, and total and differential white blood cell counts. RESULTS: The study stresses the significant increase of TNF-α, IL-8, and IL-10 in animal breeders, with the highest values in pig breeders, and a slight but statistically significant increase in albumin and total serum proteins. CONCLUSIONS: The findings of the presented study suggest a condition of immune system activation in animal breeders, with the highest levels observed in pig breeders. These changes may be attributable to exposure to organic dusts, endotoxins, or to the different biological agents present in the rural environment. The prognostic significance of these findings, however, remains unclear, but the observed changes might be indicative of a risk of developing respiratory toxic and allergic diseases, which need to be further investigated.

Farmers' exposure to herbicides in North Italy: assessment under real-life conditions in small-size rice and corn farms.

Although rice and corn are two main cash crops in Lombardy (North Italy) and their cultivation involves several thousands of farmers, risk assessment of pesticide exposure is rarely done, especially in small and medium sized enterprises. With the use of pads for environmental monitoring (OECD protocol), of pre- and post-exposure 24h urine collection for biological monitoring and of hand wash for hands' exposure, we measured the exposure of 28 agricultural workers to propanil and terbuthylazine in real-life working conditions. In propanil applicators, median daily exposure on the clothes was 73.5µmol per worker, while the exposure on the skin was 22.4µmol. For terbuthylazine, these exposures were 37.2µmol and 0.86µmol per worker, respectively. Median excretion of the propanil metabolite (3,4-dichloroaniline) after exposure was 84nmol in 24h urine, and 13nmol for the metabolite of terbuthylazine. Risk assessment performed by comparing to the AOELs of the applied active ingredients with an estimated internal dose, obtained based on the measured levels of skin and hand exposure and the percentage of dermal absorption of the active ingredients considered showed that 4 propanil workers, and no terbuthylazine workers, were overexposed. Our study helps define and confirm relationships between different exposure determinants, which can be used in the development of tools for risk assessment of exposure to pesticides in small and medium sized enterprises.

Integration of biological monitoring, environmental monitoring and computational modelling into the interpretation of pesticide exposure data: introduction to a proposed approach.

Open field, variability of climatic and working conditions, and the use of complex mixtures of pesticides makes biological and environmental monitoring in agriculture, and therefore risk assessment and management, very complicated. A need of pointing out alternative risk assessment approaches, not necessarily based on measures, but simple, user-friendly and reliable, feasible also in the less advanced situations and in particular in small size enterprises, arises. This aim can be reached through a combination of environmental monitoring, biological monitoring and computational modelling. We have used this combination of methods for the creation of "exposure and risk profiles" to be applied in specific exposure scenarios, and we have tested this approach on a sample of Italian rice and maize herbicide applicators. We have given specific "toxicity scores" to the different products used and we have identified, for each of the major working phases, that is mixing and loading, spraying, maintenance and cleaning of equipment, the main variables affecting exposure and inserted them into a simple algorithm, able to produce "exposure indices". Based on the combination of toxicity indices and exposure indices it is possible to obtain semiquantitative estimates of the risk levels experienced by the workers in the exposure scenarios considered. Results of operator exposure data collected under real-life conditions can be used to validate and refine the algorithms; moreover, the AOEL derived from pre-marketing studies can be combined to estimate tentative biological exposure limits for pesticides, useful to perform individual risk assessment based on technical surveys and on simple biological monitoring. A proof of principle example of this approach is the subject of this article.

A simple and fast method for the determination of selected organohalogenated compounds in serum samples from the general population.

Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) have been widely used in industry and agriculture. Due to their persistence and bioaccumulation, they were globally spread in the environment and may still be found in environmental and biological media, despite the international restrictions on production and use. The main aim of our study was to develop a simple and fast method suitable for the establishment of the reference values for 15 PCB congeners and 16 OCPs in general population subgroups. A cost- and time-saving screening procedure using gas chromatography coupled with low-resolution mass spectrometry, was improved and validated before application to the analysis of real samples. The overall method was validated including uncertainty measurement. Preliminary field data were collected from 95 volunteers living in two Italian areas. HCB, p,p'-DDE, PCB 153, PCB 138 and PCB 180 were the most frequently detected compounds. Age and residence area were found to be significant variables for the most abundant compounds, while no correlation between serum concentrations and gender was observed. Our results suggest that long-banned substances, including PCBs and the pesticides HCB and DDT's breakdown product, are still detectable in the general population.

Intention to leave nursing in a major Milan hospital: current situation and future perspectives.

BACKGROUND: European nurses manifest a significant tendency to leave nursing, with Italy in second place after the United Kingdom. It is therefore necessary to address the problem in national and local settings and to identify possible areas of intervention. OBJECTIVES: This paper discusses the results of a survey carried out in a major hospital in Milan (Italy) aimed at finding solutions to the above problem. METHODS: In 2004 a questionnaire was administered to the entire nursing staff. Associations between intention to quit, socio-demographic variables and job characteristics were verified by means of Mann-Whitneys's and Kruskall-Wallis' tests, Spearman's correlation coefficients and logistic regression. RESULTS: Nearly 60% of nurses considered leaving, at least every now and then. The tendency to quit was associated with job dissatisfaction, burnout symptoms and the job market situation. For those for whom nursing had a moral value, the tendency to quit was less marked. Characteristics such as regularity of shifts, type of ward, type of contract and number of working hours did not show any statistically significant association with tendency to quit. CONCLUSIONS: There are many organizational aspects that may be of importance in influencing nurses' intention to leave the profession. Efforts are needed to re-define the role of nurses in healthcare organization, adjusting salaries to the actual cost of living and preventing conditions that could lead to psychological exhaustion. Mentorship could also be useful for a better integration of nurses in hospital wards.

A study of the glutathione metaboloma peptides by energy-resolved mass spectrometry as a tool to investigate into the interference of toxic heavy metals with their metabolic processes.

To better understand the fragmentation processes of the metal-biothiol conjugates and their possible significance in biological terms, an energy-resolved mass spectrometric study of the glutathione conjugates of heavy metals, of several thiols and disulfides of the glutathione metaboloma has been carried out. The main fragmentation process of gamma-glutamyl compounds, whether in the thiol, disulfide, thioether or metal-bis-thiolate form, is the loss of the gamma-glutamyl residue, a process which ERMS data showed to be hardly influenced by the sulfur substitution. However, loss of the gamma-glutamyl residue from the mono-S-glutathionyl-mercury (II) cation is a much more energetic process, possibly pointing at a strong coordination of the carboxylic group to the metal. Moreover, loss of neutral mercury from ions containing the gamma-glutamyl residue to yield a sulfenium cation was a much more energetic process than those not containing them, suggesting that the redox potential of the thiol/disulfide system plays a role in the formal reduction of the mercury dication in the gas phase. Occurrence of complementary sulfenium and protonated thiol fragments in the spectra of protonated disulfides of the glutathione metaboloma mirrors the thiol/disulfide redox process of biological importance. The intensity ratio of the fragments is proportional to the reduction potential in solution of the corresponding redox pairs. This finding has allowed the calculation of the previously unreported reduction potentials for the disulfide/thiol pair of cysteinylglycine, thereby confirming the decomposition scheme of bis- and mono-S-glutathionyl-mercury (II) ions. Finally, on the sole basis of the mass spectrometric fragmentation of the glutathione-mercury conjugates, and supported by independent literature evidence, an unprecedented mechanism for mercury ion-induced cellular oxidative stress could be proposed, based on the depletion of the glutathione pool by a catalytic mechanism acting on the metal (II)-thiol conjugates and involving as a necessary step the enzymatic removal of the glutamic acid residue to yield a mercury (II)-cysteinyl-glycine conjugate capable of regenerating neutral mercury through the oxidation of glutathione thiols to the corresponding disulfides.

Electrospray ionization and triple quadrupole tandem mass spectrometry study of some biologically relevant homo- and heterodimeric cadmium thiolate conjugates.

A series of 19 compounds of general formula R1S-Cd-SR2, R1, and R2, being some biologically relevant thiol amino acids and peptides, were prepared by direct reaction of cadmium(II) ions and thiols in water at millimolar concentration. The obtained products were characterized by electrospray ionization and triple quadrupole tandem mass spectrometry. The source spectra of stoichiometric 1:2 Cd-thiol systems containing either an individual thiol or equimolar mixtures of two different thiols featured several Cd-containing signals, although at much lesser intensity than in the previously reported experiments with mercury(II) (J. Am. Soc. Mass Spectrom. 2004, 15, 288-300). Also, the relative intensity of the homo- and heterodimeric thiolates were significantly different from the theoretically expected 1:2:1 ratio, thus pointing at some degree of discrimination between the different thiols. In particular, homo-cysteine showed much less reactivity than cysteine, and penicillamine and cysteine methyl ester much less than the free amino acid. The fragment spectra show structure-specific ions for the different ligands bound to the metal ion and allow a stand-alone determination of the connectivity also of isomeric pairs. The fragmentation pathways are similar to those observed for the corresponding mercury(II) analogues, with the addition of further intense and specific fragments, one formally carrying a Cd-bound OH ligand and one connected as a five-membered oxazolone carrying a cadmium-bis-thiolate side chain, both formed with a high intensity. Energy-resolved fragmentation data show that metal-free ions can be generated from cysteine but not from glutathione conjugates and point to the possibility of unveiling differences in the biochemical behavior of the conjugates of different heavy metals through the detailed study of their mass spectrometric fragmentation.

Assay of urinary alpha-fluoro-beta-alanine by gas chromatography-mass spectrometry for the biological monitoring of occupational exposure to 5-fluorouracil in oncology nurses and pharmacy technicians.

The validation of an analytical method for the measurement of the unnatural amino acid alpha-fluoro-beta-alanine (AFBA), the main metabolite of the antineoplastic drug 5-fluorouracil (5FU), in urine for the biological monitoring of the exposure of hospital workers to the drug when preparing the therapeutical doses and administering to cancer patients is described. The method employed a two-step extractive derivatization of the analyte from urine to the N-trifluoroacety-n-butyl ester derivative and detection by selected-ion monitoring gas chromatography-mass spectrometry of structurally specific fragments. The limit of detection was 20 ng/mL with quantification accuracy better than +/-20% and precision (CV%) better than +/-20% in the range 0.020-10 microg/mL. Norleucine was used as the internal standard and the sample-to-sample analysis time was less than 15 min. The validated method has been applied to the biological monitoring of some hospital workers potentially exposed to 5FU and to matched control subjects. On a total number of 65 analyzed urine samples from control and exposed subjects, only three, obtained from exposed subjects, were found to be positive, with values of 20, 30 and 1150 ng/mL, respectively.

Reference values for ethylenethiourea in urine in Northern Italy: results of a pilot study.

This study was carried out to define reference values for urinary ethylenethiourea (ETU) in the Northern Italy population and to identify the sources of exposure. Ninety-five healthy subjects were selected. A spot urine sample was collected in the morning, and analyzed using GC/MS in the EI/SIM mode. Thirty-nine subjects showed urinary ETU concentrations lower than the limit of detection (LOD, 0.4 microg/g creatinine), and the remainders ETU concentrations ranging from 0.5 to 11.6 microg/g creatinine. No correlation was shown between smoke or alcohol intake and urinary ETU concentrations. Based on data on ethylene-bis-dithiocarbamate (EBDC) concentrations in food, we estimated a total EBDCs intake of 31.7-50.1 microg/day. These values are largely below the ADIs, but explain the presence of small amounts of ETU in the urine samples we have analyzed. Finally, it was estimated that the mean ETU in urine in the Italian general population is 0.6-0.8 microg/g creatinine, with a 95th percentile of 4.5-5.0 microg/g creatinine. These values can be used as reference, to compare the results of biological monitoring activities carried out on EBDCs occupationally and environmentally exposed populations.

Characterization of the disulfides of bio-thiols by electrospray ionization and triple-quadrupole tandem mass spectrometry.

Glutathione and other intracellular low molecular mass thiols act both as the major endogenous antioxidant and redox buffer system and, as recently highlighted, as an important regulator of cellular homeostasis. Such cellular functions are mediated by protein thiolation, a newly recognized post-translational modification which involves the formation of mixed disulfides between GSH and key disulfide-linked Cys residues in the native protein structure. It is also well known that thiol-seeking heavy metals, such as mercury, cadmium and lead, may interfere in this regulatory system, thus disrupting the cellular functioning. To identify such mixed disulfides in order to investigate their biological role, 15 homo- and heterodimeric disulfides were prepared by air oxidation of binary mixtures containing cysteine, homocysteine, penicillamine, N-acetylcysteine, N-acetylpenicillamine and glutathione and their protonated molecules were characterized by mass spectrometry. Collisionally activated unimolecular decomposition of protonated homo- and heterodimeric disulfides generated by electrospray ionization gives rise to fission of the disulfide system both between the two sulfur atoms and across the C--S bonds, to yield structurally specific fragments which allow one to define the structure of the compounds and to discriminate between isomeric compounds. Fission between the sulfur atoms yields a pair of R--S(+) ions and, in some cases, also the complementary fragments corresponding to the protonated amino acids. Fission across the C--S bonds mainly occurs in the disulfides of N-acetylcysteine and N-acetylpenicillamine and gives rise to non-S-containing fragments formally similar to those obtained from some mercapturic acids. The complementary fragments, formally connected as R--S--S(+) ions are also observed. Fragmentation of glutathione disulfides mainly shows the characteristic loss of the terminal gamma-linked glutamic acid and little, if any, fragmentation of the disulfide system.

Molecular characterization of homo- and heterodimeric mercury(II)-bis-thiolates of some biologically relevant thiols by electrospray ionization and triple quadrupole tandem mass spectrometry.

A series of 24 compounds of general formula R(1)S-Hg-SR(2), R(1) and R(2) being biologically relevant thiol-containing amino acids and peptides (cysteine, homo-cysteine, penicillamine, N-acetyl-cysteine, N-acetyl-penicillamine, cysteinyl-glycine, gamma-glutamyl-cysteine and glutathione) were prepared by direct reaction of mercury(II) ions and thiols in water at millimolar concentration. The obtained products were characterized by electrospray ionization and triple quadrupole tandem mass spectrometry. The source spectra of equimolar mixtures of two different thiols reacting with a stoichiometric amount of mercury(II) show the peak clusters of the three theoretically expected bis-thiolato-mercury(II) complexes with relative intensities close to the theoretically expected 1:2:1 ratio, thus pointing at lack of substantial discrimination between the different thiols, the only observed exception being homo-cysteine, which is less reactive than cysteine and penicillamine. The fragment spectra are structure-specific for the different ligands bound to the metal ion and allow a stand-alone discrimination of some constitutional isomer pairs. Among the peculiar fragmentation processes observed, loss of neutral ammonia from protonated symmetrical and unsymmetrical mercury(II)-bis-thiolates with free, protonizable amino groups leads to the formation of thiirane-carboxylic bound species; this process is suppressed when the protonated amino group is in the gamma-position with respect to the sulfur atom, as in the case of compounds with homo-cysteine. This unusual behavior may hint at unforeseen mechanisms for the interaction of mercury(II) with biological structures, ultimately leading to cellular and organ toxicity. Compounds with N-acetylated amino acids show distinctive fragment ions to which the connectivity of a protonated 2-methyl-oxazoline-5-carboxylic acid may be proposed on the basis of the loss of water and of the elements of formic acid. Finally, the adducts of mercury(II) with glutathione and gamma-glutamyl-cysteine feature a distinctive decomposition channel by loss of a pyroglutamic unit, much the same as protonated glutathione, glutathione disulfide, the S-glutathionyl adducts of biologically occurring electrophiles and other (pseudo)-peptides with gamma-glutamyl bonds.

[Evolution of industrial toxicology toward vanishing doses and the human genome]. / Evoluzione della tossicologia industriale tra dosi effimere e genoma umano.

BACKGROUND: This article aims to discuss the influence that the application of the recent discoveries in genomics will have on the theory and practice of industrial toxicology in developed post-industrial countries. It is stressed that the recent advances in toxicogenomics can be integrated into the existing wealth of knowledge on the toxic properties of industrial chemicals to improve the efficacy of prevention of toxicological risk. METHODS AND RESULTS: The understanding of the biochemical and physiological mechanisms underlying susceptibility or resistance to the toxic effects of industrial xenobiotics, and in particular to carcinogens, allows us to split the epidemiologically derived relationship linking the frequency of disease in the exposed population to the level of workplace contamination into a set of sequential sub-relationships linking: a) the exposure level to that of workplace contamination; b) the internal dose to the exposure level; c) the biological effect (e.g., measured through biochemical markers of early effect) to the internal dose; d) the frequency of disease to that of observation of early biochemical effects. Each of the cited relationships is affected by a degree of uncertainty due to the variability of biological response among the examined individuals, which in turn requires a definition of the statistical limits for the association functions between the variables. As a consequence, the possibility of investigating the individual biochemical and physiological steps in the causal mechanism that links toxic exposure to disease does not necessarily lead to an increase in the information potential of biological monitoring, since the uncertainty due to inter-individual variability is amplified through the sequence of causal relationships to the point that the data from biological monitoring become valueless with regard to the prediction of the frequency or probability of disease. This is particularly true when exposure to 'low doses' is investigated, as is now increasingly frequent in post-industrial developed countries, where workplace contamination is now greatly reduced to levels which may be borderline with those in the general environment. Thus at the low-dose end of the range of contamination and exposure values there is an area where, for statistical reasons consequent to the heterogeneity of examined populations, a quantitative prediction of internal exposure due to environmental contamination, of biological adverse effects due to exposure levels and of frequency of disease due to the extent or frequency of biological effects is no longer reliably possible. This in turn impairs the preventive efficacy of biological monitoring. CONCLUSIONS: A closer integration between industrial toxicology and state-of-the-art molecular genetics derived from the recent sequencing of the human genome is the way to overcome the limitations described. In particular, the individual subjects in the examined populations can be classified with regard to some genetically controlled characters relevant to the biotransformation of xenobiotics and to DNA repair and the statistical analysis of data can be performed on more homogeneous subpopulations, in order to decrease inter-individual variability of biochemical and physiological response. This in turn increases the predictive power of the biological markers, both of dose and effect, and improves the efficacy of prevention, e.g., by highlighting oversensitive subpopulations or lifestyles which can increase the risk of occupational and environmental disease.