RESUMO
Paraoxonase 1 (PON1) is an A-esterase calcium-dependent enzyme that is associated with high-density lipoprotein (HDL) and capable of hydrolyzing a wide variety of substrates, including organophosphate (OP) pesticides. The PON1 phenotype can be modulated by multiple internal and external factors, thereby affecting the catalytic capacity of the enzyme. The aim of this study was to evaluate factors that could modulate PON1 activity in a sample occupationally exposed to pesticides. A cross-sectional, descriptive, and analytical study was carried out with 240 workers. The participants were stratified according to their level of pesticide exposure as reference, moderate-exposure, and high-exposure groups. PON1 activities (arylesterase/AREase, CMPAase, and ssPONase (salt-stimulated)) were determined by spectrophotometry, and the Q192R and L55MPON1 genotypes by real-time PCR. The most frequent genotypes were heterozygous (QR) and homozygous (LL) for PON1Q192R and PON1L55M polymorphisms, respectively. The internal factors associated with the activity of PON1 were the PON1 genotypes (55 and 192) and biochemical parameters related to the lipid profile, in contrast, various external factors related to diet and harmful habits as well as with exposure to pesticides were associated with the activity of PON1. However, using a multivariate mixed ordinal regression model, we found a significant reduction of ssPONase activity in the high-exposure group compared with the reference group only in haplotypes QQLL and RRLL.
Assuntos
Arildialquilfosfatase/genética , Hidrolases de Éster Carboxílico/química , Compostos Organofosforados/química , Praguicidas/química , Arildialquilfosfatase/química , Arildialquilfosfatase/metabolismo , Estudos Transversais , Genótipo , Humanos , Exposição Ocupacional , Fenótipo , Polimorfismo GenéticoRESUMO
Fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate) is a racemic organophosphorus nematicide widely used in agriculture around the world. The paraoxonase 1 from human serum (PON1) is a phosphotriesterase (PTE) that hydrolyses several xenobiotics including drugs and organophosphorus compounds (OPs). In this work, the separation of the enantiomers of fenamiphos by HPLC using the column CHIRALCEL OJ and a mobile phase of hexane/ethanol (99/1) is presented. A liquid-liquid extraction method was implemented for the characterization of commercial nematicide hydrolysis by PON1 Q192R alloforms of human serum from children and adults. The results show a recovery of 94% for each isomer from the biological matrix. The method resulted linear response in a range concentration between 50 and 800µM with a detection and quantification limit between 0.6 and 2µM for the (+)-fenamiphos, and between 0.7 and 2.3µM for the (-)-fenamiphos. The levels of the Ca(2+)-dependent hydrolysis (residual concentration [µM]) quantified during 30min of reaction were only just 4-14% for both fenamiphos enantiomers with the three alloforms of PON1 Q192R of the two groups of serum studied. These results demonstrate that human serum PON1 is could be involved in the detoxification of a limited number of organophosphorus insecticides.
Assuntos
Antinematódeos/química , Arildialquilfosfatase/química , Compostos Organofosforados/química , Adulto , Criança , Humanos , Hidrólise , Isoenzimas/químicaRESUMO
The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F), and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK(a) calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolylmethylphosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK(a) calculations for the substrate-bound and unbound enzyme showed a significant pK(a) shift from standard values (ΔpK(a) = ±3 units) for residues His254 and Arg275. MD simulations of protonated His254 revealed a dynamic hydrogen bond network connecting the catalytic residue Asp301 via His254 to Asp232, Asp233, Arg275, and Asp235, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between Asp301 and His254 were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue His254, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the open to the closed substate promoting a tighter binding of paraoxon.