Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolases and Phosphoric Triester Hydrolases

Anspaugh, Douglas D.; Roe, R. Michael

doi:10.1002/0471140856.tx0410s13

“… D. japonica has PON1 activity . Quantification of PTEH activity (Anspaugh and Roe 2002) from adult wild-type D. japonica planarians. The specificity of the activity was determined by the addition of 1 µM EDTA to inhibit the calcium-dependent activity of PON1.…”

Section: Resultsmentioning

confidence: 99%

“… D. japonica has CES activity that is distinct from ChE activity. a) Quantification of CEH activity (Anspaugh and Roe 2002) from homogenates of wild-type adult D. japonica . CEH activity was inhibited by 10 mM methyl paraoxon.…”

Section: Resultsmentioning

confidence: 99%

Bioactivation and detoxification of organophosphorus pesticides in freshwater planarians shares similarities with humans

Ireland

¹

,

Rabeler

²

,

Gong

³

et al. 2022

Preprint

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Organophosphorus pesticides (OPs) are a chemically diverse class of insecticides that inhibit acetylcholinesterase (AChE). Many OPs require bioactivation to their active oxon form via cytochrome P450 to effectively inhibit AChE. OP toxicity can be mitigated by detoxification reactions performed by carboxylesterase and paraoxonase. The relative extent of bioactivation to detoxification varies among individuals and between species, leading to differential susceptibility to OP toxicity. Because of these species differences, it is imperative to characterize OP metabolism in model systems used to assess OP toxicity to adequately evaluate potential human hazard. We have shown that the asexual freshwater planarian Dugesia japonica is a suitable model to assess OP neurotoxicity and developmental neurotoxicity via rapid, automated testing of adult and developing organisms in parallel using morphological and behavioral endpoints. D. japonica has two cholinesterase enzymes with intermediate properties between AChE and butyrylcholinesterase that are sensitive to OP inhibition. Here, we demonstrate that this planarian contains the major OP metabolic machinery to be a relevant model for OP neurotoxicity studies. Adult and regenerating D. japonica can bioactivate chlorpyrifos and diazinon into their respective oxons. Significant AChE inhibition was only observed after in vivo metabolic activation but not when the parent OPs were directly added to planarian homogenate. Additionally, we found that D. japonica has both carboxylesterase and paraoxonase activity. Using specific chemical inhibitors, we show that carboxylesterase activity is distinct from cholinesterase activity. Taken together, these results further support the use of D. japonica for OP toxicity studies.

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“…PON1 activity was determined in vitro using an adapted version of the phosphoric triester hydrolase (PTEH) assay described in (Anspaugh and Roe 2002). Wild-type intact planarians were homogenized as described above but in cold 100 mM Tris-Cl, pH 8.…”

Section: Paraoxonase-1 (Pon1) Activity Assaysmentioning

confidence: 99%

“…To determine the specificity of the reaction, 2 µl of 0.5 mM EDTA was pre-incubated with 1 ml of the homogenate for 10 minutes before the PTEH activity. Since PON1 requires calcium for its enzymatic activity (Anspaugh and Roe 2002), EDTA was used as a negative control to inhibit PTEH activity.…”

Section: Paraoxonase-1 (Pon1) Activity Assaysmentioning

confidence: 99%

Bioactivation and detoxification of organophosphorus pesticides in freshwater planarians shares similarities with humans

Ireland

¹

,

Rabeler

²

,

Gong

³

et al. 2022

View full text Add to dashboard Cite

Organophosphorus pesticides (OPs) are a chemically diverse class of insecticides that inhibit acetylcholinesterase (AChE). Many OPs require bioactivation to their active oxon form via cytochrome P450 to effectively inhibit AChE. OP toxicity can be mitigated by detoxification reactions performed by carboxylesterase and paraoxonase. The relative extent of bioactivation to detoxification varies among individuals and between species, leading to differential susceptibility to OP toxicity. Because of these species differences, it is imperative to characterize OP metabolism in model systems used to assess OP toxicity to adequately evaluate potential human hazard. We have shown that the asexual freshwater planarian Dugesia japonica is a suitable model to assess OP neurotoxicity and developmental neurotoxicity via rapid, automated testing of adult and developing organisms in parallel using morphological and behavioral endpoints. D. japonica has two cholinesterase enzymes with intermediate properties between AChE and butyrylcholinesterase that are sensitive to OP inhibition. Here, we demonstrate that this planarian contains the major OP metabolic machinery to be a relevant model for OP neurotoxicity studies. Adult and regenerating D. japonica can bioactivate chlorpyrifos and diazinon into their respective oxons. Significant AChE inhibition was only observed after in vivo metabolic activation but not when the parent OPs were directly added to planarian homogenate. Additionally, we found that D. japonica has both carboxylesterase and paraoxonase activity. Using specific chemical inhibitors, we show that carboxylesterase activity is distinct from cholinesterase activity. Taken together, these results further support the use of D. japonica for OP toxicity studies.

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“…Microbial degradation of organic pollutants is generally considered to be caused by enzymes. Phosphoric triester hydrolases (EC 3.1.8), the most studied chlorpyrifos-degrading enzymes, include methyl parathion hydrolase (MPH), OP acid anhydrolase (OPAA), OP degradation enzyme (OPD), phosphotriesterase (PTE) and glycerophosphodiesterase (GPD) [23,24]. All these enzymes belong to the metalloenzymes class and have a bivalent cationic bimetallic activity center.…”

Section: Introductionmentioning

confidence: 99%

Rapid Biodegradation of the Organophosphorus Insecticide Chlorpyrifos by Cupriavidus nantongensis X1T

Shi

¹

,

Fang

²

,

Qin

³

et al. 2019

IJERPH

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Chlorpyrifos was one of the most widely used organophosphorus insecticides and the neurotoxicity and genotoxicity of chlorpyrifos to mammals, aquatic organisms and other non-target organisms have caused much public concern. Cupriavidus nantongensis X1T, a type of strain of the genus Cupriavidus, is capable of efficiently degrading 200 mg/L of chlorpyrifos within 48 h. This is ~100 fold faster than Enterobacter B-14, a well-studied chlorpyrifos-degrading bacterial strain. Strain X1T can tolerate high concentrations (500 mg/L) of chlorpyrifos over a wide range of temperatures (30–42 °C) and pH values (5–9). RT-qPCR analysis showed that the organophosphorus hydrolase (OpdB) in strain X1T was an inducible enzyme, and the crude enzyme isolated in vitro could still maintain 75% degradation activity. Strain X1T can simultaneously degrade chlorpyrifos and its main hydrolysate 3,5,6-trichloro-2-pyridinol. TCP could be further metabolized through stepwise oxidative dechlorination and further opening of the benzene ring to be completely degraded by the tricarboxylic acid cycle. The results provide a potential means for the remediation of chlorpyrifos- contaminated soil and water.

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Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolases and Phosphoric Triester Hydrolases

Cited by 3 publications

References 15 publications

Bioactivation and detoxification of organophosphorus pesticides in freshwater planarians shares similarities with humans

Bioactivation and detoxification of organophosphorus pesticides in freshwater planarians shares similarities with humans

Bioactivation and detoxification of organophosphorus pesticides in freshwater planarians shares similarities with humans

Rapid Biodegradation of the Organophosphorus Insecticide Chlorpyrifos by Cupriavidus nantongensis X1T

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