Comparative study of esterase activities in different tissues of marine fish species Trachurus trachurus, Merluccius merluccius and Trisopterus luscus.

Pesticides are one of the most frequently anthropogenic xenobiotics detected in water. Among these, the organophosphorus pesticides (OPs) are very widely used in agriculture due to their broad spectrum of activity and their low price, but they also have high potent effects as neurotoxic compounds in non-target organisms. The aim of this study was to evaluate biomarkers acetylcholinesterase (AChE), butyrylcholinesterase (BChE), propionylcholinesterase (PChE) and carboxylesterase (CbE) in the representative Atlantic fish species Trachurus trachurus, Merluccius merluccius and Trisopterus luscus from "Rías Gallegas", a traditional Spanish fishing area. These esterase activities were evaluated in the brain, muscle and liver to determine the most adequate tissue to measure such enzymatic activities. The sensitivity of AChE and CbE activities from different tissues the widely used organophosphorus insecticide chlorpyrifos (CP), and its toxic metabolite (CP-oxon) was also tested. AChE activity was predominant in all tissues of the analysed species (particularly in brain constituting from 78.33%, 89.83% and 88.43% of total ChEs in Trachurus trachurus, Merluccius merluccius and Trisopterus luscus, respectively). Under in vitro exposure, esterases were shown to be highly sensitive to CP and especially to CP-oxon. Moreover, a similar effect observed on AChE and CbE activities could suggest that CbE activity might contribute efficiently against the toxic effects of CP, especially in muscle and the liver. The presence of BChE, PChE and upper CbE activities in muscle and the liver and their OP-sensibilities can be used to study their function in the pesticide biochemical detoxification pathways with a prominent role as a safeguarding mechanism against pesticide toxicity.

Detección de siete micotoxinas tricotecenos en cereales infantiles mediante extracción QuEChERS y cromatografía líquida espectrometría de masas en tándem / Detection of seven trichothecene mycotoxins in infant cereal foods by QuEChERS extraction and liquid chromatography coupled to tandem mass spectrometry

Las micotoxinas tricotecenos se encuentran comúnmente en los cereales, como el trigo, la cebada, el maíz, la avena, el centeno y los productos derivados. Los bebés y los niños pequeños se consideran un grupode alto riesgo debido al gran consumo de alimentos a base de cereales en relación con su peso corporal. En este estudio se ha desarrollado y validado un método rápido, selectivo y sensible para la cuantificación simultánea de 7 micotoxinas tricotecenos (HT-2 toxina, T-2, diacetoxiscirpenol (DAS), deoxinivalenol (DON), 3-acetil-DON, 15-acetil DON, y fusarenon-X (FUS-X) en cereales infantiles. Las micotoxinas se han extraído de las muestras mediante QuEChERS modificado (acrónimo de rápido, fácil, barato, eficaz, robusto y seguro). El método se basa en una única extracción con una mezcla de metanol y acetonitrilo, seguido de una etapa de extracción/partición después de la adición de la sal, y un paso de limpieza utilizando extracción en fase sólida de dispersión (EPS-D). El análisis se llevó a cabo usando cromatografía líquida combinada con un espectrómetro de masas en tándem de triple cuadrupolo (EM/EM) con ionización por electrospray en modo positivos (ESI+) con monitorización de reacción múltiple (MRM). El método validado demostró tener una buena exactitud (>72% de recuperación), reproducibilidad (<10% de desviación estándar relativa interdia) y sensibilidad para las micotoxinas seleccionadas (los límites de detección del método oscilaron entre 0,02 mg kg-1 a 0,15 mg kg-1). Se analizaron 57 muestras de cereales infantiles comercializadas en España revelando la presencia de FUS-X, 15-acetil DON, 3-acetil-DON y T-2 en 9 (15,5%) muestras (AU)

Trichothecene mycotoxins are commonly found in cereals, including wheat, barley, maize, oats, rye and derived products. Infants and young children are considered the highest risk group because their large dietary intake of cereal-based foods in relation to their body weight. In this study, a rapid, selective and sensitive method was developed and validated for the simultaneous quantification of 7 trichothecenes (HT-2 toxin, T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), 3-acetyl-DON, 15-acetyl-DON, and fusarenon-X (FUS-X) in infant cereal food. Mycotoxins have been extracted from the samples using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure. The method was based on a single extraction with a mixture methanol and acetonitrile, followed by an extraction/partitioning step after the addition of salt, and a cleanup step utilizing dispersive solid-phase extraction (D-SPE). The analysis was carried out using a liquid chromatography combined with a triple–quadrupole tandem mass spectrometer (MS/MS) by electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). The validated method showed to be accurate (>72% recovery), reproducible (<10% relative standard deviation interday) and sensitive for the selected mycotoxins (method detection limits ranged from 0.02 mg kg-1 to 0.15 mg kg-1). The screening of 57 samples of infant cereal food commercialized in Spain revealed the presence of FUS-X, 15-Acetyl-DON, 3-Acetyl- DON and T-2 in 9 (15.5%) samples (AU)

Brain acetylcholinesterase, malondialdehyde and reduced glutathione as biomarkers of continuous exposure of tench, Tinca tinca, to carbofuran or deltamethrin.

In this study, the chronic effect of the insecticides carbofuran and deltamethrin on acetylcholinesterase (AChE) activity and malondialdehyde (MDA) and reduced glutathione (GSH) levels were examined in the brain of tench. Both pesticides were evaluated in two separate experiments, and animals were exposed in a continuous flow-system to three different concentrations of carbofuran (0, 10 and 100 microg/L) and deltamethrin (0, 0.0039 and 0.039 microg/L) for 60 days. After that period, animals were kept into pesticide-free water for other 30 days. In all cases, animals were sampled every 10 days all along the experience. AChE activity was significantly inhibited in fish exposed to 100 microg/L of carbofuran, during the first 30 days of exposition, returning to basal levels after this initial period. With respect to deltamethrin exposure, AChE activity was not significantly affected. When considering MDA levels, significant changes could only be detected during the recovery period for both pesticides, with a maximum of induction at 70 and 80 days, respectively associated to the highest dose of carbofuran and deltamethrin. Similarly, GSH levels varied all along the experience, with a maximum of significant increase at day 80 of exposition to the highest dose of both pesticides. This study shows that changes in AChE brain activity in tench can be used as a biomarker of early pesticide exposition in environmental monitoring programs, whereas MDA and GSH levels could be more associated to long-term expositions. The above results confirm and broaden former observations, suggesting that more investigations are needed before these biochemical parameters can be used as biomarkers.

210Pb and stable lead content in fungi: its transfer from soil.

The uptake and transfer of natural radionuclides, other than (40)K, from soil to mushrooms has been somewhat overlooked in the literature. Their contribution to the dose due to the consumption of mushrooms was considered negligible. But the contribution of (210)Pb in areas unaffected by any recent radioactive fallout has been found to be significant, up to 35% of the annual dose commitment in Spain. More than 30 species of mushrooms were analyzed, and the (210)Pb detected was in the range of 0.75-202 Bq/kg d.w. A slight difference was observed between species with different nutritional mechanisms (saprophytes > or = mycorrhizae). The (210)Pb content was correlated with the stable lead content, but not with its predecessor in the uranium radioactive series, (226)Ra. This suggested that (210)Pb was taken up from the soil by the same pathway as stable lead. The bioavailability of (210)Pb in soil was determined by means of a sequential extraction procedure (NH(4)OAc, 1M HCl, 6M HCl, and residue). About 30% of the (210)Pb present in the soil was available for transfer to mushrooms, more than other natural radionuclides in the same ecosystem. Lycoperdon perlatum, Hebeloma cylindrosporum, and Amanita curtipes presented the highest values of the available transfer factor, ATF. As reflected in their ATF values, the transfer from soil to mushroom of some natural and anthropogenic radionuclides was in the following order:

Cytotoxicity of paraquat in freshly isolated rat hepatocytes: effects of L-carnitine.

The effects of L-carnitine, a mitochondrial carrier of fatty acids, on paraquat (PQ) cytotoxicity in freshly isolated rat hepatocytes were studied. Addition of PQ (10 mM) to hepatocytes resulted in a time-dependent depletion of intracellular glutathione (GSH) accompanied by an increase in accumulation of malondialdehyde (MDA) in the incubation medium which proceeded to a loss of cell viability. Pretreatment of hepatocytes with L-carnitine (50-mM) alone did not affect cell viability or intracellular levels of GSH, or accumulation of MDA in the medium during the incubation period; however, pretreatment with L-carnitine 30 min prior to PQ addition did promote the depletion of intracellular GSH and MDA accumulation induced by PQ, and ultimately enhanced the cytotoxicity of PQ.

Accentuation of paraquat-induced toxicity by L-carnitine in mice.

The effects of L-carnitine on toxicity induced by paraquat (PQ) in mice were investigated. L-carnitine pretreatment surprisingly promoted the toxicity of PQ in a dose-dependent manner shortening the survival time. The maximum effect occurred when L-carnitine, at a dose of 16 mmol/kg, was intraperitoneally administered 30 min before an intraperitoneal injection of PQ (75 mg/kg). Lipid peroxidation in lung significantly increased 6 h after PQ administration. L-carnitine accelerated this effect since L-carnitine-pretreated mice already showed a significant increase of lung malondialdehyde 1 h after PQ administration. In liver, PQ administration did not produce lipid peroxidation; nevertheless L-carnitine-pretreated mice showed a significant increase of malondialdehyde 6 h after PQ administration. Lung and liver glutathione decreased in mice receiving only PQ but this effect was not significantly changed by L-carnitine pretreatment. These results indicate that L-carnitine accelerates PQ-induced mortality rate by facilitating lipid peroxidation.

Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1.

The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01-0.5 mM), and at higher concentrations by silybin. Cell damage by t-butyl hydroperoxide was also prevented by silymarin and silybin, but less efficiently. However, the covalent binding of the acetaminophen intermediate, formed via P450 2E1, was unaffected by addition of the flavonoids. Silybin did not influence microsomal 2E1-catalyzed demethylation of N-nitrosodimethylamine. Neither did demethylation of N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the in vivo administration of silybin. Addition of silymarin or silybin to primary cultures of isolated hepatocytes did not prevent cell damage induced by exposure to the P450 2E1 substrate CCl4. In contrast, the mere presence of low concentrations (25-50 microM) of these compounds was found to inhibit cell attachment to the matrix and eventually resulted in cell damage. We conclude that contrary to earlier reports we found no evidence for an interaction of silymarin or silybin with cytochrome P450 2E1. This suggests that the antioxidant and free radical scavenging properties may account for most of the therapeutic effect of these compounds. The untoward effect of silymarin on cultured cells may have consequences when considering long-term prescription of this therapeutic agent.

The effects of silymarin on the attachment and viability of primary cultures of rat hepatocytes.

The effects of the hepatoprotective compound silymarin on hepatocytes in primary culture were studied. Exposure of cells in primary culture (both conventional cells and perivenous or periportal cells isolated by the digitonin-collagenase perfusion technique) to high concentrations of silymarin surprisingly demonstrated that silymarin per se was cytotoxic. Incubation of cells for 18 hr with silymarin at concentrations exceeding 25 mum abruptly increased cell damage, whereas viability decreased in a more linear fashion with increasing concentrations of its major constituent, silybin. Morphologically, cell cultures exposed to silymarin concentrations lower than 20 mum appeared normal, but at higher concentrations intercellular contacts were lost; cells appeared granulated and took up eosin. Silymarin and silybin at these doses were also found to prevent cell attachment. The mechanism responsible for this effect at relatively low concentrations of silymarin during prolonged exposure in the primary cell culture system is not clear at present. The effects of low doses on cell attachment to the matrix suggest an action on the cell membrane and/or on the cytoskeleton.

Comparative study of the hepatoprotective effect of silymarin and silybin on isolated rat hepatocytes.

The flavonoid silymarin and its main active component silybin have been used in the treatment of toxic liver diseases. In order to evaluate the hepatoprotective potency of both these compounds, their effects on the viability, lipid peroxidation and reduced glutathione (GSH) depletion induced by allyl alcohol (AA) and tert-butyl hydroperoxide (t-BuOOH) in suspensions of isolated hepatocytes were investigated. Cells were preincubated for 30 min with silymarin and silybin before the addition of AA or t-BuOOH. Samples were taken after 1-2 hr of incubation. Cell death, after 2 hr of incubation with 0.2 mm AA, was prevented by 0.01 mm silymarin; however, 2 mm silybin was required to give comparable protection. The presence of silymarin reduced AA-induced lipid peroxidation by more than 90%, whereas silybin was much less effective. The near-complete depletion of intracellular GSH by AA was restored in a dose-dependent manner by silymarin, but silybin did not have this effect. Protection by silymarin against the toxic effects of t-BuOOH was less pronounced than that against AA. In conclusion, silymarin was much more effective than silybin in preventing the toxic effects induced by two pro-oxidant toxins, AA and t-BuOOH.

Effect of L-carnitine on ketone bodies, redox state and free amino acids in the liver of hyperammonemic mice.

L-Carnitine stimulates urea synthesis in mice given a LD100 of ammonium acetate. Unprotected mice show decreased levels of hepatic ketone bodies and lowered NADH/NAD+ ratio in both cytosol and mitochondria. L-Carnitine enhances markedly the production of beta-hydroxybutyrate and raises the NADH/NAD+ ratio in mitochondria. The alterations induced by ammonium acetate in the free amino acid pool are prevented by L-carnitine. The results shown in this paper indicate that L-carnitine stimulates fatty acid oxidation as well as flux through the Krebs cycle in hyperammonemic mice and that these effects may be responsible for the increase in urea synthesis in these animals.

Effect of hyperammonemia on the levels of carnitine in mice.

Decreased carnitine levels have been noted in conditions of hyperammonemia. We have measured carnitine and its derivatives in acute and sustained hyperammonemia in mice and studied the effect of carnitine administration thereon. Sustained hyperammonemia decreased carnitine in liver and muscle. Acetylcarnitine was decreased in liver and muscle in both acute and sustained hyperammonemia but increased in brain. Long-chain acylcarnitines decreased in brain and muscle in acute hyperammonemia and in liver and muscle is sustained ammonia intoxication. Intraperitoneal administration of carnitine increased the levels of free carnitine and acyl derivatives, especially in liver, but sustained hyperammonemia significantly affected the distribution of exogenous carnitine. The importance of these findings relative to the alterations of lipid metabolism observed in Reye's syndrome and inherited hyperammonemias, as well their implication in the protective effect of carnitine on hyperammonemia, are discussed.

Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice.

L-Carnitine protects mice against acute ammonia intoxication. The effect of L-carnitine on in vivo incorporation of [14C] bicarbonate into urea has been investigated in mice given large doses of ammonium acetate. The hepatic content of N-acetylglutamate has been measured. Following ammonia administration the animals given L-carnitine have much higher production of urea than the unprotected mice. The marked protective effect of L-carnitine on ammonium acetate-induced hyperammonemia and on the increase in urea synthesis is not due primarily to activation of N-acetylglutamate synthetase.