A.J. García-Fernández

Morphological characterisation of BGM (Buffalo Green Monkey) cell line exposed to low doses of cadmium chloride.

Morphological changes in the Buffalo Green Monkey (BGM) cell line after exposure to a subcytotoxic dose (0.062 mM, equivalent to EC(10)-effective concentration 10%) of cadmium chloride have been evaluated. Cells were exposed for 24 h and the effects observed at the ultrastructural level by transmission and scanning microscopy. Using transmission electron microscopy, the most notable findings in treated cells were the presence of intranuclear inclusion bodies and thin intracytoplasmic granules associated to myelin figures and the presence of apoptotic bodies. Other morphological alterations included cell vacuolisation and a reduced cytoplasm volume, condensation of the mitochondria and a decreased number of cytoplasmic organelles, except lysosomes and autophagic vacuoles, which increased in number. Scanning electron microscopy pointed to a cell with a disrupted perinuclear region and a decrease in the number of surface microvilli. We conclude that the BGM cell line may be considered an useful tool for toxicological studies involving cadmium.

Cadmium- and lead-induced apoptosis in mallard erythrocytes (Anas platyrhynchos)

Cadmium, lead and cadmium-lead (1:10) induced apoptosis were studied using mallard blood cells. The allowable range in concentrations were: 0.01-0.5, 0.1-5.0, and 0.01:0.10-0.50:5.00 mM, for cadmium, lead and cadmium-lead, respectively. The lowest EC(50) achieved was for cadmium (0.22+/-0.04 mM). Two doses from each treatment group were chosen to study apoptosis and the presence of metals in cells. The percentage of apoptotic cells increased as the concentration of metals increased. The percentage of cells with intracellular metals was high for both exposure levels and the quantity of intracellular metal was greater for exposure to high concentrations. Morphological alterations for all types of exposure were related to the diverse range of effects that these metals have on membranes. We suggest that the decrease in the number of erythrocytes observed in specimens suffering from lead and cadmium poisoning is related to the induction of apoptosis.

Detection of strychnine by gas chromatography-mass spectrometry in the carcase of a Bonelli's eagle (Hieraaetus fasciatus).

basic compounds. The sample was homogenised, acidified with a solution of 35 per cent hydrochloric acid and centrifuged. The supernatant was collected for extraction using diethyl ether as the solvent, and both ethereal and aqueous phases were obtained. The aqueous phase was then alkalinised using sodium hydroxide and extracted with chloroform in order to obtain the final basic extract for analysis. The ethereal phase was used to obtain the neutral and acid extracts: first it was alkalinised using sodium bicarbonate and immediately extracted with diethyl ether to obtain neutral extract. The remaining aqueous phase was acidified with hydrochloric acid and extracted with diethyl ether in order to obtain the final acid extract. Organic solvents of the final basic (chloroform), neutral and acid (diethyl ether) extracts were then evaporated at 40°C to a 0·5 ml final volume using a gentle stream of nitrogen. The extracts were analysed by a gas chromatography-mass spectrometry (GC-MS) system, which consisted of an Agilent 5973 N mass spectrometer and Agilent 6890 N gas chromatograph. The capillary column used was a HP-5MS. The carrier gas was helium and its flow rate was 1 ml/minute. The oven temperature was increased from 60°C (held for one minute) to 310°C (held for two minutes) at 10°C/minute. Ionisation was performed with the electron impact at 70 eV. Three chromatograms were run, for the acid, neutral and basic extracts. Mass spectra corresponding to all chromatographic peaks were compared with those included in the NBS75K.L spectrum library. Analytical results of the extracts from animals used as controls were all negative. Two strychnine isomers were detected in the neutral extract from the Bonelli’s eagle carcase; they had retention times of 14·63 and 14·75 minutes, respectively (Fig 1). Comparison of the mass spectrum with the NBS75K.L library revealed 99 per cent similarity with strychnine (Fig 2). A third peak, at 15·69 minutes, also showed 50 per cent similarity with strychnine. Marques and others (2000) also detected two isomers of strychnine using GC-MS; however, the extraction and purification procedures used by these authors were specific for strychnine and, therefore, more laborious and expensive. In the period 1994 to 1996, strychnine was the most frequent toxin used in poisoned baits in south-east Spain (Maria-Mojica and others 1998); in the last review produced by the authors’ laboratory, strychnine and aldicarb occupied the first positions in the ranking of poisoned baits (MotasFIG 1: Chromatographic peaks of strychnine in extracts of tissue samples taken from the decomposed carcase of a Bonelli’s eagle (Hieraaetus fasciatus) A bu nd an ce ( m V ) 250,000

In vitro evaluation of cell death induced by cadmium, lead and their binary mixtures on erythrocytes of Common buzzard (Buteo buteo).

Cadmium and lead are persistent and ubiquitous metals that can cause several deleterious effects in living beings. Apoptosis and necrosis are two types of cell death that can be found after in vivo and in vitro exposure to these metals. In this study, isolated red blood cells from living captive Common buzzard (Buteo buteo) were exposed in vitro to different concentrations of lead, cadmium, and the mixture lead-cadmium in a proportion of 1:10 (similar to that found in previous field studies). Data obtained from dose-response curves were used to evaluate the interactive effects of metal mixtures on cell viability. In general, except for the exposure to NOEC, additivity was the most frequently observed response. As described in human, after in vitro exposure, lead was highly accumulated in buzzard erythrocytes, while cadmium accumulation was scarce. Finally, the type of cell death (apoptosis or necrosis) induced by the exposure to different concentrations of these heavy metals and their mixtures was evaluated in the red blood cells. Apoptosis was found to be the main type of cell death observed after cadmium and/or lead exposure. However, this exposure caused an increase in lysis or necrosis, especially if red blood cells were exposed to high doses.

Validation of a QuECheRS method for analysis of neonicotinoids in small volumes of blood and assessment of exposure in Eurasian eagle owl (Bubo bubo) nestlings

Neonicotinoid pesticides have gained great interest in the last years both for agricultural and domestic use. Since the information on their environmental distribution or the effects derived from exposure to ecosystems and biota is scarce, new analytical techniques are being developed for monitoring studies. In this sense, two extraction techniques based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology to detect the neonicotinoids authorised in Spain (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, nitenpyram and thiamethoxam) were adapted and compared: a) using acetate buffer (AB); and b) using citrate buffer (CB). For detection and quantification, high performance liquid chromatography coupled with time of flight mass spectrometry (HPLC/TOF-MS) was used. The CB method provided a wide range of recoveries (68-134%) and accuracy (4-9%). The AB method provided good recoveries (59-76%, 59% corresponded to clothianidin) precision (4-11%) linearity (0.987-0.998%) and limit of quantification (2-10ng/mL) for all the compounds. To test the effectiveness of the technique, we analysed 30 blood samples of free-ranging nestlings of Eurasian eagle owl (Bubo bubo). The only compound detected, in one nestling from a dry land farming area, was imidacloprid, with a concentration of 3.28ng/mL. To our knowledge, this is the first study of neonicotinoid pesticides in free-ranging birds of prey using non-destructive samples, providing the first insight for biomonitoring studies. Further studies, including toxicokinetics and toxicodynamics, are recommended to assess the risk for these species.

Estrogenic activity of zearalenone, α-zearalenol and β-zearalenol assessed using the E-screen assay in MCF-7 cells

Abstract Mycotoxins, including zearalenone (ZEA), can occur worldwide in cereals. They can enter the food chain and cause several health disorders. ZEA and its derivatives (α-zearalenol, α-ZOL and β-zearalenol, β-ZOL) have structural analogy to estrogen, thus they can bind to estrogen receptors (ERs). In order to characterize the estrogenic activity of ZEA, α-ZOL and β-ZOL, the proliferation of ER-positive human breast cancer cells (MCF-7) exposed to these mycotoxins was measured. After exposure at levels ranging from 6.25 to 25 µM, cell proliferation was evaluated by using the E-Screen bioassay. In accordance with previous studies, our results show the estrogenic activity of ZEA, α-ZOL and β-ZOL in MCF-7 cells. This effect is related to ZEA and its metabolites being flexible enough to bind to mammalian ERs. The relative proliferative effect (RPE) ranged from 10% to 91%. The α-ZOL induced the highest proliferative effect due to its higher affinity for the ERs compared to the other mycotoxins.

Haematocrit and blood biochemical parameters in free-living Eurasian eagle owls (Bubo bubo) from Southeastern Spain: study of age and sex differences

The study of haematocrit and biochemical parameters is considered a useful tool for diagnosis of health status in birds. However, an adequate interpretation requires reference values for each species. Moreover, factors such as age and sex should also be taken into account, as it is well known that they can also influence several parameters. For these reasons, serum biochemical parameters (albumin, calcium, phosphorus, aspartate aminotransferase, alkaline phosphatase, cholesterol, triglycerides, creatine kinase, γ-glutamyltransferase, glucose, lactate dehydrogenase, total proteins and uric acid) were studied in 15 adults (6 males and 9 females) and 258 chicks of free-living Eurasian eagle owl (Bubo bubo), one of the owls with the largest distribution range worldwide. Most blood clinical parameters were higher in nestlings than in adults, but only significantly for alkaline phosphatase, phosphorus and uric acid. On the contrary, γ-glutamyltransferase was significantly lower in nestlings. In adults, sex differences were not significant. Haematocrit was studied in 13 adults (2 males and 11 females) and 162 nestlings, and levels were significantly higher in adults than in nestlings. Because samples were obtained from healthy free-living individuals and the values fall within the range of levels considered physiological for similar species, we suggest that can be used as reference values for Eurasian eagle owls.