Lourdes Rodrigo

Validation of a method to quantify chromium, cadmium, manganese, nickel and lead in human whole blood, urine, saliva and hair samples by electrothermal atomic absorption spectrometry.

For biological monitoring of heavy metal exposure in occupational toxicology, usually whole blood and urine samples are the most widely used and accepted matrix to assess internal xenobiotic exposure. Hair samples and saliva are also of interest in occupational and environmental health surveys but procedures for the determination of metals in saliva and hair are very scarce and to our knowledge there is no validation of a method to quantify Cr, Cd, Mn, Ni and Pb in four different human biological materials (whole blood, urine, saliva and axilary hair) by electrothermal atomization atomic absorption spectrometry (ETAAS). In the present study, quantification methods for the determination of Cr, Cd, Mn, Ni and Pb in whole blood, urine, saliva and axilary hair were validated according to the EU common standards. Pyrolisis and atomization temperatures have been determined. The main parameters evaluated were: detection and quantification limits, linearity range, repeatability, reproducibility, recovery and uncertainty. Accuracy of the methods was tested with the whole blood, urine and hair certified reference materials and recoveries of the spiked samples were acceptable ranged from 96.3 to 107.8%.

Immunohistochemical evidence for the expression and induction of paraoxonase in rat liver, kidney, lung and brain tissue. implications for its physiological role

Studies on the localization of paraoxonases (PONs) are of interest because of its involvement in both the detoxication of activated organophosphorus pesticides and in the prevention of peroxidative damage to phospholipids and cholesteryl-esters in LDL and HDL particles and cell membranes during the atherogenic process. In the present study, we have investigated the cellular localization of PON1 by immunohistochemistry in different rat tissues. The protein was mainly detected in the endothelial lining of every tissue studied (liver, kidney, lung and brain). Besides, it was found in hepatocytes from the centrolobular region of the liver, in the glomeruli and basal pole of the proximal convoluted tubule of the kidney, in cells from bronchiolar epithelium and type I pneumocytes of the lung, and in leptomeningeal cells, ependymal cells and ventricular side of choroid plexus cells of the brain. However, neurons and glia lacked immunostaining. After 3-methylcholanthrene induction an increase in the intensity of immunostaining was observed in the same areas, as well as an additional staining in midzonal hepatocytes. On the basis of the tissue distribution observed for PON1, it is proposed that this enzyme might have a function related to the inactivation of oxidative stress by-products (either at a cellular level or blood-vessel wall) and other environmental chemicals. At present it has not yet been established whether the paraoxonase detected in the various tissues is truly a product of the PON1 gene or could represent products of the PON2 or PON3 genes.

Paraoxonase activity and genetic polymorphisms in greenhouse workers with long term pesticide exposure

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL) associated protein, which plays a critical role in the pathogenesis of atherosclerosis, although it was primarily associated with the hydrolysis of organophosphorus compounds. PON1 was initially thought to be independent from physiological or pathological states, although recently some environmental factors have been reported to modulate its activity. In this study, we have investigated the promoter (PON1-108C/T and-909 C/G) and coding region (PON1 192Q/R and 55L/M) polymorphisms, as well as PON1 activity towards different substrates (paraoxon, phenylacetate and diazoxon) in 102 individuals with long term low dose exposure to pesticides in a plastic greenhouse setting (sprayers), who are probably the group of agricultural workers with the highest exposure to pesticides. PON1 activity towards paraoxon was nonsignificantly decreased (up to 53.5%) in the sprayers subgroup exposed to organophosphates (n-41) compared with nonsprayers acting as controls (n-39). None of the genotypes studied was associated significantly with the subgroup of individuals exposed to organophosphates, although differences between sprayers and nonsprayers were observed in the PON1-909 G/C polymorphism. Among the environmental factors that significantly predicted lower rates of PON1 activity towards paraoxon are, interestingly, the exposure to organophosphates and current smoking. By contrast, the utilization of protective clothing while spraying pesticides inside the greenhouses was positively associated with PON1 activity, very likely by preventing the pesticides from being absorbed. This study suggests that chronic exposure to pesticides might decrease PON1 activity and pinpoints the potential usefulness of monitoring PON1 activity in occupational settings where exposure to organophosphates occurs.

Effect of Long-Term Exposure to Pesticides on Plasma Esterases from Plastic Greenhouse Workers

Previous reports in animals consideredβ-glucuronidase activity as a novel biomarker of anticholinesterase (organophosphates and carbamates) pesticides exposure. Acid phosphatase activity was also shown to increase after organophosphates exposure. In addition, there is evidence that the paraoxonase status influences sensitivity to specific pesticides. In this study, activities ofβ-glucuronidase, acid phosphatase, cholinesterase, and paraoxonase were measured in plasma from plastic greenhouse workers exposed over the long term to different pesticides, including organophosphates and carbamates, in order to evaluate the potential chronic toxicity of pesticides at occupational level. Our results show that activities of paraoxonase and cholinesterase were decreased in applicators of pesticides compared to non-applicators. Likewise, it was found that activities ofβ-glucuronidase and acid phosphatase were associated with pesticide exposure in humans, and that both biochemical parameters were related to each other. Interestingly, the paraoxonase B allele (phenotyped in plasma) was associated with a higher risk of inhibition of cholinesterase activity above a 25% level, which supports the hypothesis that paraoxonase phenotypes are associated with susceptibility of humans to anticholinesterase pesticides toxicity.

Human liver paraoxonase (PON1): Subcellular distribution and characterization

The subcellular localization and different biochemical properties of a human hepatic microsomal enzyme that hydrolyses paraoxon (paraoxonase, PON1) were studied and compared to the paraoxon hydrolase activity found in human plasma as well as in rat liver and plasma. Having evaluated the influence of the postmortem interval by a parallel experiment performed in rats, we conclude that the paraoxonase activity was preferentially localized in the microsomal fraction. The enzyme reaction was optimized according to temperature, pH, buffer, ionic strength, substrate concentration, and enzyme protein concentration. The characterization of human liver paraoxonase included the study of optimum pH, pH stability, heat inactivation assays, and kinetic parameters (Km and Vmax). In addition, the enzyme activity showed an absolute requirement for exogenous calcium. The activity was lost after incubation with EDTA and partially restored by the addition of calcium; however, other metals assayed were not able to activate the human liver enzyme as did calcium. Our results support the possible identity between human plasma and liver paraoxonases. In spite of the technical difficulties of this study and the possible interference of the postmortem changes in the results, this article represents the first systematic approach to the characterization of human liver paraoxonase.

Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties.

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability.

Interaction between human serum esterases and environmental metal compounds

Paraoxonase-1 (PON1) and cholinesterase (BChE) are two of the major human serum esterases. Although most of variation in PON1 activity results from genetic factors, there is growing evidence that environmental chemicals also modulate its activity. The aim of this study was to investigate whether environmental exposure to metal compounds has any influence on those esterases. A cross-sectional study was conducted in a representative sample of the general population of Andalusia, South of Spain. PON1 activity against different substrates (paraoxon, phenylacetate, diazoxon and dihydrocoumarin) and BChE were measured in serum from 536 healthy subjects. Potential associations of these esterases with metal compounds, age, sex and body mass index as well as life-style habits (smoking, alcohol drinking and food habits) were explored. Multiple linear regression analysis showed that blood lead levels were significantly associated with increased PON1 in serum regardless of the substrate used for the assay. Mercury also showed a significant and direct association with PON1 towards paraoxon and phenylacetate. In turn, cadmium and zinc levels were significantly associated with a decreased PON1 activity (zinc was associated with all PON1 activities and cadmium with PON1 towards paraoxon and diazoxon). Arsenic, nickel and manganese failed to be significantly associated with any of the PON1 activities assayed. PON1 192R alloform predicted significantly higher levels of arsenic and lead. BChE, however, was inversely associated with serum levels of manganese and zinc. These results suggest that PON1 and BChE activities are modulated by background exposure to metal compounds, which may have implications in public health given the defensive role played by both enzyme proteins against environmental toxicants. The potential underlying mechanisms merit further investigation.

Hair testing for cocaine and metabolites by GC/MS: criteria to quantitatively assess cocaine use

A simple, rapid and sensitive method has been developed and validated for the determination of cocaine and its main metabolites (benzoylecgonine and cocaethylene) in human hair. The method involved solid‐phase extraction with an Oasis HLB extraction cartridge and subsequent analysis by GC/MS. The limit of detection was 0.01 ng mg−1 for cocaine, 0.04 for benzoylecgonine and 0.03 for cocaethylene. The method validation included linearity (with a correlation coefficient >0.99 over the range 0.2–50 ng mg−1), intra‐ and inter‐day precision (always lower than 12%) and accuracy (mean relative error always below 17%) to meet the bioanalytical acceptance criteria. The procedure was further applied to 40 hair samples from self‐reported cocaine users arrested by the police who provided a positive urine‐analysis for cocaine, and was demonstrated to be suitable for its application in forensic toxicology. New approaches were raised to detect false‐negative results that allow a better interpretation of hair testing results. Copyright

Identification of two rat liver proteins with paraoxonase activity: biochemical evidence for the identity of paraoxonase and arylesterase

The existence of two or more enzyme forms with paraoxonase activity has been reported in sheep, rabbit, human and rat serum and recently in mouse and rat liver. In this study we describe the presence of two peaks with paraoxonase activity (M1 and M2) after non-specific affinity chromatography of rat liver microsomes on Cibacron Blue 3GA. The first peak (M1) was obtained during the washing of the column and coeluted with albumin. The second active peak (M2) was eluted with 1 M NaCl. The characterization of each peak was determined by SDS/PAGE electrophoresis and Western-blotting. A comparison of both active fractions on the basis of kinetic parameters, heat inactivation and pH stability, calcium requirement and inhibition by EDTA and several metals was performed. Our results support the fact that two proteins capable of hydrolyzing paraoxon are present in rat liver microsomes. Furthermore, during the purification to homogeneity of rat liver paraoxonase we have performed a study of its hydrolytic ability against three different substrates: paraoxon, phenylacetate and phenyl thioacetate (Paraoxonase (PON), Arylesterase (ArE), Phenyl thioacetate esterase (PTase)). The elution profile in different chromatographic steps, as well as the activity ratios from the crude extract throughout the purification process, heat inactivation and effect of inhibitors were used as identity criteria for the three hydrolytic activities. Our results show evidence for the hydrolysis of paraoxon and phenylacetate by the same protein from rat liver (paraoxonase).

Rapid determination of quetiapine in blood by gas chromatography–mass spectrometry. Application to post-mortem cases

A simple, fast and sensitive method for the determination of quetiapine in human blood has been developed and validated. The method involved a basic liquid–liquid extraction procedure and subsequent analysis by gas chromatography–mass spectrometry, previous derivatization with bis(trimethylsilyl)‐trifluoro‐acetamide and chorotrimethylsilane (99 : 1). The methods of validation included linearity with a correlation coefficient > 0.99 over the range 0.02–1 µg ml–1, intra‐ and interday precision (always < 12%) and accuracy (mean relative error always < 12%) to meet the bioanalytical acceptance criteria. The limit of detection was 0.005 µg ml–1. The procedure was further applied to post mortems from the Institute of Legal Medicine, University of Santiago de Compostela. Copyright