Federica Aureli

Oral, short-term exposure to titanium dioxide nanoparticles in Sprague-Dawley rat: focus on reproductive and endocrine systems and spleen.

Abstract The study explored possible reproductive and endocrine effects of short-term (5 days) oral exposure to anatase TiO2 nanoparticles (0, 1, 2 mg/kg body weight per day) in rat. Nanoparticles were characterised by scanning electron microscopy (SEM) and transmission electron microscopy, and their presence in spleen, a target organ for bioaccumulation, was investigated by single-particle inductively coupled plasma mass spectrometry and SEM/energy-dispersive X-ray. Analyses included serum hormone levels (testosterone, 17-β-estradiol and triiodothyronine) and histopathology of thyroid, adrenals, ovary, uterus, testis and spleen. Increased total Ti tissue levels were found in spleen and ovaries. Sex-related histological alterations were observed at both dose levels in thyroid, adrenal medulla, adrenal cortex (females) and ovarian granulosa, without general toxicity. Altered thyroid function was indicated by reduced T3 (males). Testosterone levels increased in high-dose males and decreased in females. In the spleen of treated animals TiO2 aggregates and increased white pulp (high-dose females) were detected, even though Ti tissue levels remained low reflecting the low doses and the short exposure time. Our findings prompt to comprehensively assess endocrine and reproductive effects in the safety evaluation of nanomaterials.

Changes in Selenium Speciation Associated with Increasing Tissue Concentrations of Selenium in Wheat Grain

Wheat (Triticum aestivum) collected in the Nawanshahr-Hoshiarpur Region (Punjab, India) showed the highest selenium concentrations ever recorded in cereal grains (29-185 microg g(-1)). There was a strong positive relationship between the selenium content in shoots and that in kernels, showing that grain selenium concentration can be predicted from that in the vegetative tissues of the plant. The identity and content of the selenocompounds in the grain samples and in wheat-based reference materials were investigated by HPLC-ICP-dynamic reaction cell-MS. Reversed-phase, cation exchange, and anion exchange HPLC were used to separate the selenium species after ultrasound-assisted enzymatic extraction with an ultrasonic probe. Selenomethionine and selenate accounted for 72-85% and 2-6% of the sum of the selenium species, respectively. The proportion of organic Se species varied with increasing Se content; namely, SeMet showed a relative reduction whereas the other organoselenium compounds increased up to 18-22% of the total chromatographed selenium. Se-methyl-selenocysteine was detected as a minor compound (0.2-0.5%) in high-Se wheat by both reversed-phase and cation exchange HPLC using retention time matching with the standard substance spiked to the sample extracts. Regular consumption of locally produced wheat-based food items may lead the population of the study area to an excessive intake of selenium. On the other hand, the large predominance of selenomethionine shows that local wheat can be a promising raw material for naturally enriched products to be used to supplement human and animal diets in low selenium areas.

Bioaccumulation Potential of Dietary Arsenic, Cadmium, Lead, Mercury, and Selenium in Organs and Tissues of Rainbow Trout (Oncorhyncus mykiss) as a Function of Fish Growth

The distribution and potential bioaccumulation of dietary arsenic, cadmium, lead, mercury, and selenium in organs and tissues of rainbow trout (Oncorhyncus mykiss Walbaum, 1792), a major aquaculture species, was studied in relation to fish growth over a period of >3 years. Fish were reared under normal farming conditions, that is, fed a standard fish food and exposed to negligible levels of waterborne trace elements. The age-related variations in the content of each trace element in gills, kidney, liver, muscle, and skin were studied through nonparametric regression analysis. A buildup of all elements in all tissues and organs was observed, but due to dilution with growth, the concentrations did not increase, except in a few cases such as cadmium and mercury in liver and kidney. In muscle tissue, the concentrations of mercury, lead, and selenium did not alter significantly with growth, whereas cadmium increased but remained at exceedingly low levels. The concentration of arsenic in muscle tissue peaked at 14 months and then decreased in adult specimens. Arsenic speciation by high-performance liquid chromatography--inductively coupled plasma mass spectrometry revealed that arsenic in muscle was almost exclusively present in the form of nontoxic arsenobetaine. Application of a mercury mass balance model gave predicted concentrations in agreement with measured ones and showed that in farmed rainbow trout the ratio of mercury concentrations in feed and in fish is about 1:1. Therefore, rainbow trout does not approach the limits established for human consumption even when reared with feed at the maximum permitted levels. These findings highlight the low bioaccumulation potential of toxic trace elements such as cadmium, lead, and mercury in rainbow trout following dietary exposure. On the other hand, selenium concentrations in muscle (about 0.2 microg g (-1) of fresh weight) show that rainbow trout may be a good source of this essential element.

Arsenic speciation in freshwater fish: Focus on extraction and mass balance

Arsenic (As) speciation in muscle tissues of freshwater fish was investigated with special emphasis on extraction yields and mass balances. For the quantification of water-soluble As compounds, samples were extracted using a 1:1 (v/v) methanol/water mixture. Various extraction parameters, such as extraction volume, number of additional extractions, intermediate and final volume in sample preconcentration were optimized so as to improve the extraction efficiency. Arsenic compounds were determined by HPLC with online ICP-MS, using both cation and anion exchange separation. The species studied were eel (Anguilla anguilla L.), flathead grey mullet (Mugil cephalus L.), chub (Leuciscus cephalus L.), and carp (Cyprinus carpio L.). Mean total As concentrations ranged from 354 microg kg(-1) dry weight (carp) to 1804 microg kg(-1) dry weight (mullet). Under optimized conditions, the percentage of total As extracted ranged from 64% for carp, to 82%, 84%, and 89% for grey mullet, eel and chub, respectively. Extraction of lipid-rich eel with n-hexane recovered some additional 3% of total As. The sizeable effect of sample matrix on HPLC retention time of some organoarsenicals in gradient elution cation exchange chromatography was exploited to achieve separation of coeluting compounds by analysing the same sample at different dilutions. The recovery of As from chromatographic columns was 101%, 102%, 103% and 104% for carp, chub, mullet and eel, respectively, indicating that no As was retained during chromatography. Arsenobetaine (AB) was the dominating As compound, but several other arsenicals, including arsenous acid (Asiii), arsenic acid (Asv), methylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), arsenocholine ion (AC), tetramethylarsonium ion (TETRA), oxo-arsenosugar-glycerol (AS1), oxo-arsenosugar-phosphate (AS2), oxo-arsenosugar-sulfate (AS4), thio-arsenosugar-phosphate (ThioAS2), and three unknown As compounds, were found. Arsenic speciation in carp was different compared to the other fish species, and a lower proportion of AB along with a high contribution of AS2 and ThioAS2 was found. DMA and TMAO were noteworthy minor compounds in eel and carp, respectively. Arsenic speciation and the chemical composition of fish muscle both appeared to affect the extraction yield.

Selenium bioaccessibility and speciation in biofortified Pleurotus mushrooms grown on selenium-rich agricultural residues

Cultivation of saprophytic fungi on selenium-rich substrates can be an effective means to produce selenium-fortified food. Pleurotus florida, an edible species of oyster mushrooms, was grown on wheat straw from the seleniferous belt of Punjab (India) and its potential to mobilize and accumulate selenium from the growth substrate was studied. Selenium concentration in biofortified mushrooms was 800 times higher compared with control samples grown on wheat straw from non selenium-rich areas (141 vs 0.17 μg Se g(-1) dry weight). Seventy-five percent of the selenium was extracted after in vitro simulated gastrointestinal digestion and investigation of the selenium molecular fractions by size exclusion HPLC-ICP-MS revealed that proteins and any other high molecular weight selenium-containing molecule were hydrolyzed to peptides and low molecular weight selenocompounds. Analysis of the gastrointestinal hydrolysates by anion exchange HPLC-ICP-MS showed that the bioaccessible selenium was mainly present as selenomethionine, a good bioavailable source of selenium, which accounted for 73% of the sum of the detected species. This study demonstrates the feasibility of producing selenium-biofortified edible mushrooms using selenium-rich agricultural by-products as growth substrates. The proposed approach can be used to evaluate whether selenium-contaminated plant waste materials harvested from high-selenium areas may be used to produce selenium-biofortified edible mushrooms based on the concentration, bioaccessibility and speciation of selenium in the mushrooms.

Arsenic speciation in wheat and wheat products using ultrasound- and microwave-assisted extraction and anion exchange chromatography-inductively coupled plasma mass spectrometry

Wheat appears to be the major contributor to the intake of inorganic arsenic in countries where the diet is not rice-based. Ultrasound- and microwave-assisted extraction of arsenic in wheat and wheat based food using different solvents or enzymes was investigated in terms of extraction yield and species stability. Four extraction procedures were selected for the study of arsenic speciation in wheat and wheat products by anion exchange HPLC-ICP-MS using a PRP-X100 column with 10 mM NH4H2PO4, 10 mM NH4NO3, and 2% CH3OH at pH 5.5 as the mobile phase. Total arsenic in the samples ranged from 8.6 to 29.8 ng g−1 dry weight. About 95% of the arsenic was found to be present in inorganic form with AsIII as the most abundant species, whereas the remainder was mainly DMA. Microwave-assisted extraction with HNO3 was the most effective in liberating the arsenic species, which were then satisfactorily recovered from the chromatographic column. The LODs achieved, i.e., 0.35–0.46 ng g−1 dry weight, were suitable for the determination of arsenic species at the low levels found in sample extracts.

Arsenic contamination of the environment-food chain: a survey on wheat as a test plant to investigate phytoavailable arsenic in Italian agricultural soils and as a source of inorganic arsenic in the diet.

Seven hundred and twenty-six samples of wheat grains from the majority of Italian agricultural areas were pooled into 141 composite samples, homogeneous with respect to geographical origin and wheat variety. The average arsenic concentration of the pooled samples was 9 ng g(-1), with a range of 2-55 ng g(-1) (dry weight basis). The spread of arsenic concentrations (coefficient of variation of 91%) was related to spatial variability associated with geochemical and environmental factors. Temporal variability was investigated by a 3-year longitudinal study on 7 wheat cultivars grown in 22 areas of central and northern Italy. Average year-to-year variation in arsenic levels was low, and the average of the coefficients of variation was 23%. These results show that mapping of phytoavailable arsenic in agricultural soils can be done by measuring arsenic concentration in representative samples of wheat grains. Arsenic speciation in the grain showed that As(III) and As(V) were the major As compounds, highlighting the importance of wheat as a source of inorganic arsenic in the diet.

Coumarin and cinnamaldehyde in cinnamon marketed in Italy: A natural chemical hazard?

Some plants that are processed into foods often contain natural substances that may be hazardous to human health. One example is coumarin, which is known to cause liver and kidney damage in rats, mice and probably humans. The main source of coumarin in the diet is cinnamon. The name ‘cinnamon’ is correctly used to refer to Ceylon cinnamon, also known as ‘true cinnamon’. However, other plant species are sometimes sold with the label of cinnamon. This is the case of Cinnamomun aromaticum (cassia). In recent years, due to its cheaper price, cassia is replacing true cinnamon in the European food market being largely used in the preparation of some kinds of sweets. Several European health agencies have recently warned against consuming high amounts of cassia due to its high content of coumarin. In this study, 34 samples of cinnamon and 50 samples of cinnamon-containing foodstuffs were collected from the Italian market. Quantitative determinations of coumarin and cinnamaldehyde were performed by high-performance liquid chromatography (HPLC) with diode array detector (DAD). The analytical method was in-house validated assessing recovery, repeatability, linearity, limit of detection (LOD) and limit of quantitation (LOQ). The results showed that about 51% of cinnamon samples consisted of cassia, 10% were probably a blend of cassia and Ceylon cinnamon, whereas only 39% were actually Ceylon cinnamon. As far as cinnamon-containing foods are concerned, the samples often exceeded the maximum level fixed in the European Flavourings Directive of 2 mg kg−1.

Speciation of vanadium(IV) and (V) in mineral water by anion exchange liquid chromatography-inductively coupled plasma mass spectrometry after EDTA complexation

HPLC with on-line ICP-MS was used for the determination of V(IV) and V(V) in natural mineral water. Cationic and anionic tetravalent and pentavalent vanadium species were converted into V–EDTA complexes, i.e.[VO(EDTA)]2− and [VO2(EDTA)]3−, which could be effectively separated on a short anion exchange column in <6 min. Pre-column EDTA complexation also prevented species interconversion as a result of loss of CO2 from samples and their exposure to atmospheric oxygen. A low-salt mobile phase (4 mM carbonate buffer and 5 mM EDTA–Na2) compatible with ICP-MS detection was used in chromatographic separations. The method was tested on real samples spanning one order of magnitude of V concentrations and encompassing a wide range of potential interfering ions and total dissolved solids, providing accurate and reproducible results. Mass balance budgets gave a mean value of 100% (range 96–107%). Recoveries from spiking experiments were in the range 98–103%. Precision as average intra-day repeatability was 1.2% and 0.9% for V(IV) and V(V), respectively. Average inter-day repeatability was 5.5% and 1.4% for V(IV) and V(V), respectively. Limits of detections were 0.16 μg L−1 for V(IV) and 0.025 μg L−1 for V(V). The interference arising by 35Cl16O was resolved chromatographically. V(V) was found to largely predominate in PET-bottled mineral water samples. Species distribution was consistent with theoretical predictions based on thermodynamic equilibria of vanadium in water. Overall, a rapid, sensitive, on-line method is provided which can be readily employed for monitoring purposes.

Comprehensive speciation of low-molecular weight selenium metabolites in mustard seeds using HPLC – electrospray linear trap/orbitrap tandem mass spectrometry

An analytical methodology based on high-resolution high mass accuracy electrospray ionization (ESI) tandem MS assisted by Se-specific detection using inductively coupled plasma mass spectrometry (ICP MS) was developed for speciation of selenium (Se) in seeds of black mustard (Brassica nigra) grown on Se-rich soil. Size-exclusion LC-ICP MS allowed the determination of the Se distribution according to the molecular mass and the control of the species stability during extraction. The optimization of hydrophilic interaction of LC and cation-exchange HPLC resulted in analytical conditions making it possible to detect and characterize over 30 Se species using ESI MS, including a number of minor (<0.5%) metabolites. Selenoglucosinolates were found to be the most important class of species accounting for at least 15% of the total Se present and over 50% of all the metabolites. They were found particularly unstable during aqueous extraction leading to the loss of Se by volatilization as methylselenonitriles and methylselenoisothiocyanates identified using gas chromatography (GC) with the parallel ICP MS and atmospheric pressure chemical ionization (APCI) MS/MS detection. However, selenoglucosinolates could be efficiently recovered by extraction with 70% methanol. Other classes of identified species included selenoamino acids, selenosugars, selenosinapine and selenourea derivatives. The three types of reactions leading to the formation of selenometabolites were: the Se-S substitution in the metabolic pathway, oxidative reactions of -SeH groups with endogenous biomolecules, and chemical reactions, e.g., esterification, of Se-containing molecules and other biomolecules through functional groups not involving Se.