Renzo Bagnati

Estimating community drug abuse by wastewater analysis.

Background The social and medical problems of drug abuse are a matter of increasing global concern. To tackle drug abuse in changing scenarios, international drug agencies need fresh methods to monitor trends and patterns of illicit drug consumption. Objective We tested a sewage epidemiology approach, using levels of excreted drug residues in wastewater, to monitor collective use of the major drugs of abuse in near real time. Methods Selected drug target residues derived from use of cocaine, opiates, cannabis, and amphetamines were measured by mass spectrometry in wastewater collected at major sewage treatment plants in Milan (Italy), Lugano (Switzerland), and London (United Kingdom). The amounts of drug residues conveyed to the treatment plants, reflecting the amounts collectively excreted with urine, were used to estimate consumption of the active parent drugs. Results Reproducible and characteristic profiles of illicit drug use were obtained in the three cities, thus for the first time quickly revealing changes in local consumption (e.g., cocaine consumption rose significantly on weekends in Milan). Profiles of local drug consumption based on waste-water measurements are in line with national annual prevalence estimates. Conclusions Patterns and trends of drug abuse in local communities can be promptly monitored by this tool, a convenient new complement to more complex, lengthy survey methods. In principle, searching the sewage for excreted compounds relevant to public health issues appears to have the potential to become a convenient source of real-time epidemiologic information.

Source, occurrence and fate of antibiotics in the Italian aquatic environment

Aim of this study was to provide an up-to-date assessment of the antibiotics contaminating the aqueous environment in Italy, for a better understanding of risks for the ecosystem and human health. Antibiotics were first listed in order of their theoretical environmental loads, then were measured in wastewater of some sewage treatment plants (STPs) and in rivers in Italy. Macrolides, particularly clarithromycin and spiramycin, and quinolones, particularly ciprofloxacin and L-floxacin/ofloxacin, were the most abundant antibiotics in untreated wastewater. Several of them were not removed in STPs and still remained in the treated wastewater, and a total estimate of 7-14 tons of active principles were discharged annually into the aqueous environment in Italy through this route. Results of the analysis of rivers in northern Italy agreed with these figures, with an average load of 5 kg/day, or about 1.8 tons/year, of antibiotics flowing in the River Po, at sampling sites covering a basin comprising about one-fifth of the Italian population. In conclusion, antibiotics, particularly macrolides and quinolones, are widespread environmental contaminants, and urban STPs are confirmed a major source of the contamination.

Mass spectrometric analysis of illicit drugs in wastewater and surface water.

Residues of illicit drugs have been recently found in urban wastewater and surface water. Their levels reflect the amount of drugs collectively excreted by consumers and can therefore be used to estimate drug abuse. An overview of the most widely used illicit drugs and of the analytical methods used for their detection in wastewater and surface water is presented here. Solid-phase extraction and high performance liquid chromatography-tandem mass spectrometry are the techniques that have been used for these investigations. Instrumental conditions and fragmentation patterns of illicit drugs and their metabolites are described.

Chemical characterization of Iraqi propolis samples and assessing their antioxidant potentials.

Propolis samples, collected from different geographical locations in Iraq (Baghdad, Dahuk, Mosul and Salah ad-Din), were analyzed and assessed for their anti-oxidant activity. Concentrations of phenolic compounds (flavonoids, phenolic acids and their esters) in propolis were estimated using high performance liquid chromatography coupled to electrospray mass spectrometry. Thirty-eight different compounds were identified and 33 of them were polyphenols. Other compounds were tentatively identified as clerodane diterpenoids, and one was considered unknown. Semi-quantitative measurements showed that phenolic acids and their esters were the predominant constituents in propolis extracts, followed by flavones and flavonols, and then flavanones and dihydroflavonols. Propolis samples were further spectrophotometrically characterized using the Folin-Ciocalteu reagent for the determination of total phenolic compounds. The free radical scavenging activities of propolis samples were also evaluated by using the 2,2-diphenyl-1-picrylhydrazyl assay. The results revealed that propolis extracts exhibited strong free radical scavenging activity.

Effect of diet on serum albumin and hemoglobin adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in humans.

2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is the most abundant heterocyclic amine formed in meat and fish during cooking and can be used as a model compound for this class of chemicals possibly involved in human carcinogenesis. Knowing the exposure to heterocyclic amines is important for establishing their role in human diseases. Serum albumin (SA) and globin (Gb) adducts were first tested as biomarkers of exposure to PhIP in male Fischer 344 rats given oral doses of 0.1, 0.5, 1 and 10 mg/kg. Blood samples were collected 24 hr after treatment and PhIP released from SA and Gb after acidic hydrolysis was analyzed by gas chromatography‐mass spectrometry or liquid chromatography‐tandem mass spectrometry. PhIP‐SA and Gb adducts increased linearly with the dose. Studies on 35 volunteers with different dietary habits exhibited that diet was a major determinant in the formation of both adducts. PhIP‐SA adducts were significantly higher in meat consumers than in vegetarians (6.7 ± 1.6 and 0.7 ± 0.3 fmol/mg SA; respectively, mean ± SE; p = 0.04, Mann‐Whitney U test). The Gb adduct pattern was quantitatively lower but paralleled SA (3 ± 0.8 in meat consumers and 0.3 ± 0.1 in vegetarians). PhIP‐SA adducts were no different in smokers and in non‐smokers. The results show for the first time that PhIP‐blood protein adducts are present in humans not given the synthetic compound. Both biomarkers appear to be suitable for assessing dietary exposure and internal PhIP dose and may be promising tools for studying the role of heterocyclic amines in the etiology of colon cancer and other diseases. Int. J. Cancer 88:1–6, 2000.

Analysis of diethylstilbestrol, dienestrol and hexestrol in biological samples by immunoaffinity extraction and gas chromatography-negative-ion chemical ionization mass spectrometry.

A method has been developed for the detection of diethylstilbestrol, together with dienestrol and hexestrol, using extraction with a single immunoaffinity column containing antibodies raised against diethylstilbestrol, followed by gas chromatography-negative-ion chemical ionization mass spectrometry. Immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was synthesized by introducing a carboxyl group into the diethylstilbestrol molecule and coupling this product to bovine serum albumin. The columns were used for immunoadsorption of diethylstilbestrol and other estrogens, after dilution of samples with phosphate buffer, and were eluted with acetone-water (95:5 v/v). A derivatization method suitable for gas chromatographic-mass spectrometric analysis of diethylstilbestrol and other estrogens was developed using pentafluorobenzyl bromide and ethanolic potassium hydroxide as reagents. The derivatives obtained were detectable at the sub-picogram level using gas chromatography with negative-ion chemical ionization mass spectrometry. Recoveries of cis- and trans-diethylstilbestrol, dienestrol and hexestrol from the immunoaffinity columns, determined after extraction from urine, plasma and buffer, ranged from 28 to 96%. The sensitivity for diethylstilbestrol in urine samples was ca. 10 ppt. The method was applied to the analysis of urine from calves given a single dose of 10 mg of diethylstilbestrol. Free and glucuronic acid conjugated diethylstilbestrol decreased with time, but their ratio was variable.

Screening of 21 pesticides in water by single extraction with C18 silica bonded phase columns and HRGC-MS

Abstract A rapid method has been developed for the simultaneous semi-quantitative analysis of 21 pesticides in ground waters at sub-ppb levels. The method has been used for the validation of new wells as sources of drinking water in a contaminated area in the North of Italy.

Increased concentrations of nitrophenols in leaves from a damaged forestal site

Abstract A class of organic micropollutants, the phytotoxic nitrophenols, were analysed in the vegetation (beech leaves), from two different alpine sites (Val Gerola and Val Masino) in the Lombardy Region (Northern Italy), using gas chromatography-mass spectrometry. Leaves from the more damaged site (Val Gerola) had higher mean total nitrophenol concentrations (313 ng/g) than the Val Masino samples (148 ng/g). Since the dinitrophenol levels in leaves are close to the concentrations exerting a phytotoxic effect in vitro, these pollutants presumably have an active role in the symptoms of tree diseases in these alpine sites.

Effects of cigarette smoking on the human urinary proteome

In this pilot study we used a proteomic approach to compare the urinary protein patterns of healthy smokers and non-smokers. Proteins were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. The relative abundance of three inflammatory proteins (S100A8, inter-alpha-trypsin inhibitor heavy chain 4, CD59) and that of two isoforms of pancreatic alpha amylase was significantly higher in smokers. Zinc-alpha-2-glycoprotein was the only protein down-regulated in smokers. Its abundance was significantly correlated with urinary glucocorticoids. Most of the proteins identified may be non-specific biomarkers of tobacco effects, since they are involved in inflammatory responses associated with several diseases. Of greater interest are the changes in abundance of pancreatic alpha amylase and zinc-alpha-2-glycoprotein, which after proper validation, might be candidate biomarkers of diseases resulting from exposure to tobacco smoke. The data also show for the first time that smoking can affect the expression profile of urinary proteins.

DNA damage induced by alachlor after in vitro activation by rat hepatocytes

We investigated the ability of alachlor to cause DNA damage by measuring single-strand breaks (SSB) in DNA, after metabolic activation by freshly isolated rat hepatocytes. Incubation of different concentrations of alachlor with rat hepatocytes led to numerous metabolites. The majority, isolated and identified by GC-MS analysis, were products arising from reactions catalyzed by the P-450 monooxygenase system, arylamidase and flavin mixed-function oxidase/cytochrome P-450 monooxygenase. The results, using freshly isolated rat hepatocytes, showed that in these conditions several potentially DNA damaging metabolites were produced; this experimental condition was used to assess DNA damage induced by the mixture of alachlor and its metabolites. The alkaline elution technique showed that at 200 microM and more clearly at 400 microM there were some small fragments that eluted in the first fraction. This fragmentation was probably due to alachlor cytotoxicity. In addition to the small DNA fragments eluting in the first fraction there were other larger DNA fragments. These DNA-SSB were most evident at the alachlor concentration of 400 microM, but also at 200 microM and 100 microM, whereas at 10 microM the DNA elution rate appeared comparable to that of controls. The results suggest that some unstable and DNA-reactive metabolites might interact with DNA causing SSB and such interaction might be important in relation to the mechanism of alachlor-induced DNA damage. However, it may not be possible to clarify whether SSB are the result of direct DNA interaction of the compound or of secondary cellular processes after chemical treatment.