Olga López

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.

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.

Implications of Paraoxonase-1 (PON1) Activity and Polymorphisms on Biochemical and Clinical Outcomes in Workers Exposed to Pesticides

Paraoxonase-1 (PON1) is known to play an important role in the individual susceptibility to environmental chemicals, particularly pesticides. The major results of our studies on biochemical and clinical end-points of workers long-term exposed to pesticides in a large intensive agriculture area from Southeast Spain are presented herein and compared with several other epidemiologic studies performed in different scenarios. In addition of being an individual marker of susceptibility, PON1 can be also considered a biological indicator of exposure to pesticides, since workers spraying these agents (chiefly OPs) showed decreased enzyme levels. Besides, long-term exposure to pesticides appears to indirectly elicit higher levels of PON1, which might be regarded as enzyme induction. On the other hand, carriers of the PON1 192R allele showed lower levels of erythrocyte cholinesterase and catalase, but a higher glutathione reductase activity. Regarding clinical outcomes, workers with the PON1 R allele had less risk of reporting a previous episode of pesticide poisoning as well as a lower risk of pesticide-related symptomatology. Exposure to low doses of pesticides which are metabolically activated in the liver seems to elicit subtle and early biochemical changes of hepatotoxicity. It is concluded that epidemiological studies addressing health or biochemical outcomes of workers occupationally exposed to pesticides should determine PON1 genotypes and phenotypes (activities), as these biomarkers may help in identifying those individuals at increased risk of developing pesticide toxicity or who are showing early effects after pesticide exposure