Organophosphorus poisoning in animals and enzymatic antidotes.

Organophosphorus compounds (OPs) are neurotoxic molecules developed as pesticides and chemical warfare nerve agents (CWNAs). Most of them are covalent inhibitors of acetylcholinesterase (AChE), a key enzyme in nervous systems, and are therefore responsible for numerous poisonings around the world. Many animal models have been studied over the years in order to decipher the toxicity of OPs and to provide insights for therapeutic and decontamination purposes. Environmental impact on wild animal species has been analyzed to understand the consequences of OP uses in agriculture. In complement, various laboratory models, from invertebrates to aquatic organisms, rodents and primates, have been chosen to study chronic and acute toxicity as well as neurobehavioral impact, immune response, developmental disruption, and other pathological signs. Several decontamination approaches were developed to counteract the poisoning effects of OPs. Among these, enzyme-based strategies are particularly attractive as they allow efficient external decontamination without toxicity or environmental impact and may be of interest for treatment. Approaches using bioscavengers for prophylaxis, treatment, and external decontamination are emphasized and their potential is discussed in the light of toxicological observations from various animal models. The relevance of animal models, regarding their cholinergic system and the abundance of naturally protecting enzymes, is also discussed for better extrapolation of results to human.

Enzymatic decontamination of paraoxon-ethyl limits long-term effects in planarians.

Organophosphorus compounds (OP) are highly toxic molecules used as insecticides that inhibit cholinesterase enzymes involved in neuronal transmission. The intensive use of OP for vector control and agriculture has led to environmental pollutions responsible for severe intoxications and putative long-term effects on humans and wild animals. Many in vivo models were studied over the years to assess OP acute toxicity, but the long-term effects are poorly documented. Planarian, a freshwater flatworm having a cholinergic system, has emerged as a new original model for addressing both toxicity and developmental perturbations. We used Schmidtea mediterranea planarians to evaluate long-term effects of paraoxon-ethyl at two sublethal concentrations over three generations. Toxicity, developmental perturbations and disruption of behavior were rapidly observed and higher sensitivity to paraoxon-ethyl of next generations was noticed suggesting that low insecticide doses can induce transgenerational effects. With the view of limiting OP poisoning, SsoPox, an hyperthermostable enzyme issued from the archaea Saccharolobus solfataricus, was used to degrade paraoxon-ethyl prior to planarian exposure. The degradation products, although not lethal to the worms, were found to decrease cholinesterase activities for the last generation of planarians and to induce abnormalities albeit in lower proportion than insecticides.

[Planarian, an emerging animal model for toxicology studies]. / Modèles alternatifs (6) - La planaire, un modèle animal original pour la toxicologie.

Since a few decades, a new invertebrate animal model has emerged in toxicology studies: the planarian. This non-parasitic flatworm, from phylum Platyhelminthes, has an amazing regenerative capacity and has been described as "immortal under the edge of the knife" in 1814 by Dalyell. This formidable capacity is due to the abundance of stem cells called neoblasts, allowing for a tiny fragment equivalent to 1/279th of the size of the planarian to generate a whole animal. The planarian has also a human-like nervous system with several neurotransmitters and has been used to evaluate developmental perturbations and neurotoxicity. This review summarizes the main planarian toxicology studies and highlights the potential of this original animal model for research.

Evaluation of a robust engineered enzyme towards organophosphorus insecticide bioremediation using planarians as biosensors.

Organophosphorus compounds (OPs) are neurotoxic molecules developed as insecticides and chemical warfare nerve agents (CWNAs). They are covalent inhibitors of acetylcholinesterase (AChE), a key enzyme in central and peripheral nervous systems and are responsible for numerous poisonings worldwide. Many animal models have been studied over the years but finding a suitable in vivo model to account for both acute toxicity and long-term exposure remains a topical issue. Recently, an emerging aquatic animal model harboring a mammalian-like cholinergic nervous system, the freshwater planarian from Platyhelminthes, has been used to investigate neurotoxicity and developmental disruption. Given the tremendous toxicity of OPs, various bioremediation strategies have been considered over the years to counter their poisonous effects. Among these, enzymes have been particularly highlighted as they can degrade OPs in a fast, non toxic and environmentally friendly manner. In this article we investigated the biotechnological potential for decontaminating OPs of the previously reported variant SsoPox-αsD6 from the hyperstable enzyme SsoPox, isolated from the archaea Sulfolobus solfataricus. The capacity to hydrolyze 4 new substrates (methyl-pirimiphos, quinalphos, triazophos and dibrom) was demonstrated and the degradation products generated by enzymatic hydrolysis were characterized. We further evaluated the capacity of SsoPox-αsD6 for in vivo protection of freshwater planarians Schmidtea mediterranea (Smed). The use of SsoPox-αsD6 drastically decreased mortality and enhanced mobility of planarians. Then, an enzyme-based filtration device was developed by immobilizing intact Escherichia coli cells expressing SsoPox-αsD6 into alginate beads. The efficacy of the device was demonstrated using planarians as biosensors.

Enzymatic degradation of organophosphorus insecticides decreases toxicity in planarians and enhances survival.

Organophosphorus insecticides (OPs) are toxic compounds used for agricultural purposes and responsible for severe types of contamination worldwide. OPs may also induce chronic deleterious effects and developmental disruption. Finding remediation strategies is a major concern to diminish their impact on environment and human health. Enzymes have emerged as a promising eco-friendly route for decontaminating OPs. The enzyme SsoPox from the archaea Sulfolobus solfataricus has been particularly studied, considering both its tremendous stability and phosphotriesterase activity. However, the toxicity of the degradation products generated through enzyme hydrolysis has been poorly investigated. To address both neurotoxicity and developmental perturbation, freshwater planarians from Platyhelminthes were considered to evaluate the impact of OP and degradation product exposure. Planarians have a large proportion of stem cells that give them an unconventional capacity for regeneration. OPs were found to be highly toxic to planarians and enzyme decontamination drastically enhanced survival rate. Although not completely innocuous, the degradation products were found to be less toxic than insecticides and reduced poisoning effects by increasing NOEC values by up to eight-fold. SsoPox also limited detrimental consequences on planarian mobility and enabled them to recover a non-exposed type regeneration process suggesting that enzymatic decontamination is a promising alternative to bioremediation.

Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications.

Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. Here we investigate the biotechnological properties of the mutant SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities. We tested enzyme resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization. Bacterial secreted materials from both Gram-negative and positive bacteria were harmless on SsoPox-W263I activity and could reactivate heat-inactivated enzyme. SsoPox showed resistance to harsh conditions demonstrating that it is an extremely attractive enzyme for many applications. Finally, the potential of SsoPox-W263I to be active at subzero temperature is highlighted and discussed in regards to the common idea that hyperthermophile enzymes are nearly inactive at low temperatures.