Miguel A. Sogorb

Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis

The most employed insecticides for indoor and agriculture purposes belong to carbamates, pyrethroid or organophosphates. The chemical structures of these three groups correspond to carbamic, carboxylic and triphosphoric esters. Technical monographs suggest that the hydrolysis of ester bonds of carbamates and pyrethroids plays an important role in the detoxification of these compounds. However, detailed studies about enzymes hydrolysing carbamates and pyrethroids in vertebrates are not available. Certain carbamate hydrolysing activities are associated to serum albumin. Phosphotriesterases, being of an unknown physiological role, hydrolyse (in some cases stereospecifically) organophosphorus insecticides (OP). Phosphotriesterases have been found in a multitude of species, from mammals to bacteria. A phosphotriesterase activity, EDTA-resistant, has been detected in serum albumin. Phosphotriesterases in serum of mammals display polymorphisms. Phosphotriesterases offer applications in therapy of organophosphorus poisonings, in biodegradation and bioremedation of organophosphates. Similar studies should be developed with enzymes hydrolysing pyrethroids and carbamate insecticides. Such studies will improve the knowledge of the detoxification routes in non-target species and will help to design specific and safer carbamate and pyrethroid insecticides.

The role of phosphotriesterases in the detoxication of organophosphorus compounds

The enzymes that hydrolyze organophosphorus compounds are called phosphotriesterases. The presence of phosphotriesterases has been described in a variety of tissues. The physiological role of these enzymes is not known, although a clear correlation exists between the levels of phosphotriesterases and susceptibility of the species to the toxic effects of organophosphorus compounds. Thus, mammals that possess high levels of phosphotriesterases in serum and liver are more tolerant to the toxic effects of these compounds than birds and insects - these being species considered lacking of phosphotriesterases. Because most of these enzymes are not well characterized, they are usually differentiated according to their different patterns of response to activators and/ or inhibitors. Phosphotriesterases usually depend of divalent cations and therefore EDTA usually inhibits them. A peculiar EDTA-resistant phosphotriesterase has been described in serum albumin. The biotechnological and therapeutical applications of phosphotriesterases are currently subject to study.

Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S(P)- and R(P)-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R(P)-enantiomer of the sarin analogue (k(cat)=2600 s(-1)) was the preferred substrate for the wild-type PTE relative to the corresponding S(P)-enantiomer (k(cat)=290 s(-1)). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k(cat) values for the R(P)- and S(P)-enantiomers were 410 and 4200 s(-1), respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R(P)R(C)-stereoisomer of the soman analogue was the preferred substrate for PTE. The k(cat) values for the soman analogues were measured as follows: R(P)R(C,) 48 s(-1); R(P)S(C), 4.8 s(-1); S(P)R(C), 0.3 s(-1), and S(P)S(C), 0.04 s(-1). With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k(cat) values for the soman analogues were found to be as follows: R(P)R(C,) 0.3 s(-1); R(P)S(C), 0.3 s(-1); S(P)R(C), 11s(-1), and S(P)S(C), 2.1 s(-1). Prior investigations have demonstrated that the S(P)-enantiomers of sarin and soman are significantly more toxic than the R(P)-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.

Serum Albumin is as Efficient as Paraxonase in the Detoxication of Paraoxon at Toxicologically Relevant Concentrations

Human serum albumin was able to hydrolyze the organophosphorus compounds paraoxon, chlorpyrifos-oxon, and diazoxon at toxicologically relevant concentrations. Human serum displayed two paraoxon hydrolyzing activities: the so-called paraoxonase, which is associated with the lipoprotein fraction and is calcium dependent and EDTA sensitive, and the activity associated with albumin, which is EDTA resistant and sensitive to fatty acids. Human serum albumin hydrolyzed these compounds with the same relative efficacy as lipoproteins (chlorpyrifos-oxon > diazoxon > paraoxon). The capability of detoxication of activity associated with human serum albumin was similar or even higher than paraoxonase associated with lipoproteins in the case of paraoxon at concentrations as low as those noted in an acute in vivo intoxication. However, paraoxonase activity associated with lipoprotein was more effective than paraoxonase activity associated with albumin at toxicologically relevant chlorpyrifos-oxon concentrations. These results explain why mice deficient in paraoxonase associated with lipoprotein are not more sensitive to paraoxon than wild animals.

An integrated approach for detecting embryotoxicity and developmental toxicity of environmental contaminants using in vitro alternative methods

The main available alternatives for testing embryotoxicity are cellular tests with stem cells and in vitro-ex vivo tests with embryos. In cellular tests, the most developed alternative is the embryonic stem cell test, while the most developed tests involving embryos are the zebrafish and whole embryo culture test. They are technically more complex than cellular tests, but offer the advantage of determining the expectable phenotypic alteration caused by the exposure. Many efforts are currently being made, basically through proteomic and genomic approaches, in order to obtain improvements in predictivity of these tests. Development is a very complex process, and it is highly unlikely that a single alternative test can yield satisfactory performance with all types of chemicals. We propose a step-wise approach where model complexity, and consequently technical skills and economical costs, gradually increase if needed. The first level would be run short cellular assays to detect effects in early differentiation stages. The second level would involve longer cellular embryotoxicity tests to search embryotoxicants that have an effect on late differentiation stages. The third stage would consider tests with embryos because they allow the determination of hazards based on molecular and morphological alterations, and not only on differentiating cells.

Phosphotriesterase activity identified in purified serum albumins

Abstract The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 ± 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate ( p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates.

Chlorpyrifos and its metabolites alter gene expression at non-cytotoxic concentrations in D3 mouse embryonic stem cells under in vitro differentiation: considerations for embryotoxic risk assessment.

The effects of organophosphate insecticide chlorpyrifos (CPF) on development are currently under discussion. CPF and its metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TClP), were more cytotoxic for D3 mouse embryonic stem cells than for differentiated fibroblasts 3T3 cells. Exposure to 10 μM CPF and TClP and 100 μM CPO for 12 h significantly altered the in vitro expression of biomarkers of differentiation in D3 cells. Similarly, exposure to 20 μM CPF and 25 μM CPO and TClP for 3 days also altered the expression of the biomarkers in the same model. These exposures caused no significant reduction in D3 viability with mild inhibition of acetylcholinesterase and neuropathy target esterase by CPF and severe inhibition by CPO. We conclude that certain in vivo exposure scenarios are possible, which cause inhibition of acetylcholinesterase but without clinical symptoms that reach high enough systemic CPF concentrations able to alter the expression of genes involved in cellular differentiation with potentially hazard effects on development. Conversely, the risk for embryotoxicity by CPO and TClP was very low because the required exposure would induce severe cholinergic syndrome.

Serum albumins and detoxication of anti-cholinesterase agents.

Serum albumin displays an esterase activity that is capable of hydrolysing the anti-cholinesterase compounds carbaryl, paraoxon, chlorpyrifos-oxon, diazoxon and O-hexyl, O-2,5-dichlorphenyl phosphoramidate. The detoxication of all these anti-cholinesterase compounds takes place at significant rates with substrate concentrations in the same order of magnitude as expected during in vivo exposures, even when these substrate concentrations are between 15 and 1300 times lower than the recorded K(m) constants. Our data suggest that the efficacy of this detoxication system is based on the high concentration of albumin in plasma (and in the rest of the body), and not on the catalytic efficacy itself, which is low for albumin. We conclude the need for a structure-activity relationship study into the albumin-associated esterase activities because this protein is universally present in vertebrates and could compensate for reduced levels of other esterases, i.e., lipoprotein paraoxonase, in some species. It is also remarkable that the biotransformation of xenobiotics can be reliably studied in vitro, although conditions as similar as possible to in vivo situations are necessary.

A stereospecific phosphotriesterase in hen liver and brain

O-Hexyl, O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. This compound is hydrolyzed by a phosphotriesterase known as HDCPase in hen and rat plasma, liver and brain. We studied the stereospecificity of HDCPase in hen tissues and in human and rabbit plasma employing a chromatographic method for analysis and quantification of HDCP stereoisomers. Hen and human plasma HDCPases were not stereospecific. However, rabbit plasma showed a remarkable stereospecificity to S-(-)-HDCP. High levels of stereospecific HDCPase were found in the particulate fraction of hen liver, where S-(-)-HDCP is hydrolyzed faster than R-(+)-HDCP. However, in hen brain the stereospecificity was found in the soluble fraction, where R-(+)-HDCP is hydrolyzed faster than S-(-)-HDCP. It is concluded that liver particulate fraction must be the main tissue responsible for the HDCP stereospecific biotransformation in hens. In an oral administration, the steroisomer R-(+)-HDCP would survive after passing through the liver and would interact with acetylcholinesterase and neuropathy target esterase in the nervous system.

Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity in vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes: Pnpla6, Afp, Hdac7, Vegfa, and Nes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of Nes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for in vitro risk assessment embryotoxicity.