Federico Maria Rubino

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 (β-HCH) and endosulfan.

Separation methods for methotrexate, its structural analogues and metabolites

Methotrexate (MTX) is the prototype folate antagonist cytotoxic drug, employed in the therapy of solid tumors and leukaemias, and recently also as an immunosuppressive agent in organ transplantation, in the treatment of some autoimmune diseases and in the therapy of severe asthma. MTX is one of the very few antineoplastic drugs the therapeutic concentration monitoring of which is currently employed in clinical practice and can be routinely measured in biological samples by a number of different analytical techniques, among which are immunoenzymatic and chromatographic methods. Each technique has of course its own advantages in terms of sensitivity, specificity, speed, cost and level of expertise required. Along with therapeutic drug concentration monitoring and clinical pharmacology, fundamental research into the mechanism of action of antifolate drugs is still a field which requires the measurement of MTX, of its new analogues and of their metabolites in biological samples. This review summarizes the instrumental conditions and the performance of several published chromatographic methods employed to measure MTX, its metabolites and some analogues in clinical and biological research. More than 70 papers describing chromatographic assays for MTX and its metabolites have been published in the literature between 1975 and 2000. A wide array of experimental conditions for sample preparation, analyte separation and detection have been employed. According to their chemical properties, MTX, its metabolites and analogue drugs present in several biological samples (plasma, serum, saliva, urine, cerebrospinal fluid, tissue specimens) can be extracted, separated and detected under a variety of chromatographic conditions, i.e. on different stationary phases, under a wide choice of mobile phase conditions (acidic or neutral, employing ion-pair or micellar chromatography), followed by several detection techniques (UV-Vis spectrophotometry, pre- or post-column oxidation and fluorimetry, electrochemistry, mass spectrometry). Optimized methods allow simultaneous measurement within a few minutes of the plasma levels of MTX and its main metabolites at concentrations in the low-nM range. One special field which needs sensitive, fast and inexpensive methods for the detection and measurement of MTX is the monitoring of contamination in workplace environments, such as pharmaceutical industries and oncological hospital pharmacies, and in sewage waters. The measurement of the intracellular gamma-oligo-glutamate metabolites of biological folates, of MTX and of some analogue drugs is of great importance in basic pharmacological research. The existence of empirical quantitative relationships between the retention of individual oligomers under different chromatographic conditions and the number of added glutamic acid units allows identification of the metabolites even when authentic standards are not available.

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.

Measurement of surface contamination from nucleoside analogue antineoplastic drugs by high-performance liquid chromatography in occupational hygiene studies of oncologic hospital departments

Within the frame of a continuing interest in occupational hygiene of hospitals as workplaces, we describe an automated analytical method by reversed-phase high-performance liquid chromatography for the measurement of contamination from the three most important nucleoside analogue antineoplastic drugs (5-fluorouracil, 5FU; cytarabin, CYA; gemcytabin, GCA) on such surfaces as those of preparation hoods and work-benches in departmental pharmacies of oncologic departments. Our method is characterized by a short analysis time (7 min) under isocratic conditions, by the use of a mobile phase devoid of organic solvent and by high sensitivity (LOD > or = 40 micrograms/l for all compounds), adequate to detect surface contamination above a threshold of 4 micrograms/m2 for wide surfaces and of 30 micrograms/m2 for small irregular objects. We present some results from a preliminary survey study recently performed in seven oncologic departments of two large general hospitals in Milan. To exemplify the contamination levels on various surfaces (such as on handles, floor surfaces and window glass panes, even far from the preparation hood), analyte concentrations in the order of 0.03-0.06 microgram/ml, corresponding to 0.8-1.5 micrograms of 5FU were measured on telephones, of 0.02-0.6 microgram/ml (0.85-28 micrograms/m2) of CYA were measured on table boards, of 0.05-10.6 micrograms/ml (1.2-1150 micrograms/m2) of GCA on furniture and floors. Spillage fractions up to 1% of the employed ANDs (employed daily 5FU 7-13 g; CYA 0.1-7.1 g; GCA 0.2-5 g) are measured on the polyethylene-backed paper disposable cover sheet of the preparation hood.

Toxicity of Glutathione-Binding Metals: A Review of Targets and Mechanisms

Mercury, cadmium, arsenic and lead are among priority metals for toxicological studies due to the frequent human exposure and to the significant burden of disease following acute and chronic intoxication. Among their common characteristics is chemical affinity to proteins and non-protein thiols and their ability to generate cellular oxidative stress by the best-known Fenton mechanism. Their health effects are however diverse: kidney and liver damage, cancer at specific sites, irreversible neurological damages with metal-specific features. Mechanisms for the induction of oxidative stress by interaction with the cell thiolome will be presented, based on literature evidence and of experimental findings.

Biological monitoring of exposure to tebuconazole in winegrowers

Tebuconazole (TEB) is a fungicide widely used in vineyards and is a suspected teratogen for humans. The aim of this research was to identify urinary biomarkers and the best sampling time for the biological monitoring of exposure to TEB in agricultural workers. Seven vineyard workers of the Monferrato region, Piedemont, Italy, were investigated for a total of 12 workdays. They treated the vineyards with TEB for 1–2 consecutive days, one of them for 3 days. During each application coveralls, underwears, hand washing liquids and head coverings were used to estimate dermal exposure. For biomonitoring, spot samples of urine from each individual were collected starting from 24 h before the first application, continuing during the application, and again after the application for about 48 h. TEB and its metabolites TEB-OH and TEB-COOH were measured by liquid chromatography/triple quadrupole mass spectrometry. TEB contamination of coveralls and total dermal exposure showed median levels of 6180 and 1020 μg. Urinary TEB-OH was the most abundant metabolite; its excretion rate peaked within 24 h after product application (post 24 h). In this time frame, median levels of TEB-OH and TEB-COOH ranged from 8.0 to 387.8 μg/l and from 5.7 to 102.9 μg/l, respectively, with a ratio between the two metabolites of about 3.5. The total amount of urinary metabolites (U-TEBeq) post 24 h was significantly correlated with both TEB on coveralls and total dermal exposure (Pearson’s r=0.756 and 0.577). The amount of metabolites excreted in urine represented about 17% of total dermal TEB exposure. Our results suggest that TEB-OH and TEB-COOH in post-exposure urine samples are promising candidates for biomonitoring TEB exposure in agricultural workers.

Obsolete Pesticides – A Threat to Environment, Biodiversity and Human Health

Since Rachel Carson’s passionate warning in the seminal 1962 book ‘Silent Spring’, which is now celebrating 50 years from its publication, concern on the steadily increasing accumulation in the environment of chemically robust, biologically persistent and possibly toxic organochlorine pesticides led to their substitution with less threatening products and finally to stop or limit their production and to severely restrict their use. In particular, the Stockholm Convention on Persistent Organic Pollutants (POPS) signed in 2001 banned or greatly restricted 12 chlorinated organic compounds or classes due to their toxicity and ability to accumulate in the environment and to magnify through the global trophic network. Among them are 11 pesticides namely aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, hexachloro-benzene, kepone, lindane, mirex, toxaphene. These pesticides played a historical role in mitigating the health impact of parasite-borne human pathogens such as malaria parasites and in protecting food crops to allow better feeding of an increasingly raising population especially in sub-tropical and tropical areas. Concern for human and environmental health is mainly due to long-term effects of some substances, in particular through endocrine disruption, interference with reproduction, carcinogenicity, although the actual size of effects of real-life exposure is still an active and debated research topic. Risk assessment and risk-benefit analysis of some key pesticides such as DDT still need a thorough understanding of the toxicity mechanisms and of its relevance to humans in the different life-stages. As a consequence of the Stockholm ban, large stockpiles of unusable pesticides accumulate in some countries and thus present considerable threat to the environment and to human health, also due to unavoidable degradation of the active formulated substances into poorly tractable materials. To avoid environmental damage through contamination of water and agricultural land resources by leaching or improper disposal of repositories, inventories of existing stockpiles are needed to plan and carry adequately safe disposal interventions. Methods for disposal need to tackle the peculiar chemical characteristics of these highly chlorinated aliphatic and aromatic compounds, i.e., very low water solubility and an unusual stability towards acid, basic and oxidizing conditions. Research and technology development in this field is currently exploiting the most advanced ‘green chemistry’ approaches, aimed at an as complete mineralization of organo-chlorine substrates with as negligible production of toxic waste. We will draw a brief historical perspective of the genesis of this pivotal environmental problem, review the use of the ‘dirty dozen’ pesticides and their contribution to the Green Revolution improvement of agricultural food availability, the ecotoxicological and human health concern and the current efforts to front the problems raised by their use.

Health risks in international container and bulk cargo transport due to volatile toxic compounds

To ensure the preservation and quality of the goods, physical (i.e. radiation) or chemical pest control is needed. The dark side of such consents may bear health risks in international transport and production sharing. In fact, between 10% and 20% of all containers arriving European harbors were shown to contain volatile toxic substances above the exposure limit values. Possible exposure to these toxic chemicals may occur not only for the applicators but also the receiver by off gassing from products, packing materials or transport units like containers. A number of intoxications, some with lethal outcome, occur not only during the fumigation, but also during freight transport (on bulk carriers and other transport vessels), as well as in the logistic lines during loading and unloading. Risk occupations include dock-workers, seafarers, inspectors, as well as the usually uninformed workers of importing enterprises that unload the products. Bystanders as well as vulnerable consumers may also be at risk. Ongoing studies focus on the release of these toxic volatile substances from various goods. It was shown that the half-lives of the off-gassing process range between minutes and months, depending on the toxic substance, its chemical reactivity, concentration, the temperature, the contaminated matrix (goods and packing materials), and the packing density in the transport units. Regulations on declaration and handling dangerous goods are mostly not followed.It is obvious that this hazardous situation in freight transport urgently requires preventive steps. In order to improve awareness and relevant knowledge there is a need for more comprehensive information on chemical hazards and a broader implementation of the already existing regulations and guidelines, such as those from ILO, IMO, and national authorities. It is also necessary to have regular controls by the authorities on a worldwide scale, which should be followed by sanctions in case of disregarding regulations. Further, fumigated containers must have a warning sign corresponding to international recommendations and national regulations, and freight documents have to indicate any potential hazard during stripping the goods.

Measurement of S‐methylcysteine and S‐methyl‐mercapturic acid in human urine by alkyl‐chloroformate extractive derivatization and isotope‐dilution gas chromatography–mass spectrometry

S-methylcysteine (SMC) is a minor amino acid naturally excreted in human urine, a protective agent against oxidative stress and a biotransformation product of the fumigant biocide methyl bromide and of nicotine. A metabolic source of SMC is catabolism of the repair catalytic protein MGMT (EC 2.1.1.37), which specifically removes the methyl group from the modified DNA nucleotide O-6-methyl-guanine to revert the normal GC base pairing. To assess the value of SMC and of S-methylmercapturic acid (SMMA) as candidate biomarkers of proliferative phenomena, a sensitive analytical method by GC-MS was applied in a pilot study of healthy subjects to assess their urinary elimination and the intra- and inter-individual variability. Extractive alkylation with butylchloroformate-n-butanol-pyridine (Husek technique) was employed for sample derivatization and isotope dilution GC-MS with S-[CD(3) ]-SMC and -SMMA was applied for specific and sensitive detection. To resolve the target analytes from the main coeluting interferents in the derivatized urine extract a medium-polarity stationary phase was employed. SMMA was not detected in the morning urine of three healthy fertile-age women followed for one month above the minimum detectable level of approx. 500 µg/L while SMC concentrations were in the 0.02-0.7 µg/mL range (n = 61) with large inter-day and inter-individual variations. In a young healthy male urine samples taken throughout a few days yielded concentrations in the same 90-810 µg/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes.

A study on the solution and gas-phase chemistry of Mn(III) and Fe(III) tetraarylporphyrin complexes by fast-atom bombardment mass spectrometry : 1: Generation of molecular signals.

Fast-atom bombardment mass spectrometry has been used to investigate the chemical behavior of Fe(III) and Mn(III) tetraarylporphyrins (TAP) in both the condensed and gas phases and to clarify the mechanisms responsible for the production of positive and negative ions. The differences in the behavior of Fe(III) and Mn(III) complexes in the positive ion mode could be correlated with those in their electronic structures and knowledge of the mechanism for the generation of negatively charged species was applied to characterize the counterion coordinated to the Mn(III)-TAP. Thus, the unprecedented, complete characterization of even complex Mn(III)-TAP was made possible.