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ACS Applied Materials & Interfaces Jun 2024The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some...
The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, sp. 4Hβ as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of sp. 4Hβ, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.
PubMed: 38920304
DOI: 10.1021/acsami.4c06501 -
Talanta Jun 2024Methyl paraoxon (MP) is a highly toxic, efficient and broad-spectrum organophosphorus pesticide, which poses significant risks to ecological environment and human...
Methyl paraoxon (MP) is a highly toxic, efficient and broad-spectrum organophosphorus pesticide, which poses significant risks to ecological environment and human health. Many detection methods for MP are based on the enzyme catalytic or inhibition effect. But natural biological enzymes are relatively expensive and easy to be inactivated with a short service life. As a unique tool of nanotechnology with enzyme-like characteristics, nanozyme has attracted increasing concern. However, a large proportion of nanozymes lack the intrinsic specificity, becoming a main barrier of constraining their use in biochemical analysis. Here, we use a one-pot reverse microemulsion polymerization combine the gold nanoclusters (AuNCs) with molecularly imprinted polymers (MIPs), polydopamine (PDA) and hollow CeO nanospheres to synthesize the bright red-orange fluorescence probe (CeO@PDA@AuNCs-MIPs) with high phosphatase-like activity for selective detection of MP. The hollow structure possesses a specific surface area and porous matrix, which not only increases the exposure of active sites but also enhances the efficiency of mass and electron transport. Consequently, this structure significantly enhances the catalytic activity by reducing transport distances. The introduced MIPs provide the specific recognition sites for MP. And Ce (III) can excite aggregation induced emission of AuNCs and enhance the fluorescent signal. The absolute fluorescence quantum yield (FLQY) of CeO@PDA@AuNCs-MIPs (1.41 %) was 12.8-fold higher than that of the GSH-AuNCs (0.11 %). With the presence of MP, Ce (IV)/Ce (III) species serve as the active sites to polarize and hydrolyze phosphate bonds to generate p-nitrophenol (p-NP), which can quench the fluorescent signal through the inner-filter effect. The as-prepared CeO@PDA@AuNCs-MIPs nanozyme-based fluorescence method for MP detection displayed superior analytical performances with wide linearities range of 0.45-125 nM and the detection limit of 0.15 nM. Furthermore, the designed method offers satisfactory practical application ability. The developed method is simple and effective for the in-field detection.
PubMed: 38879946
DOI: 10.1016/j.talanta.2024.126434 -
Clinical Toxicology (Philadelphia, Pa.) May 2024Intentional and unintentional organophosphorus pesticide exposure is a public health concern. Organothiophosphate compounds require metabolic bioactivation by the...
INTRODUCTION
Intentional and unintentional organophosphorus pesticide exposure is a public health concern. Organothiophosphate compounds require metabolic bioactivation by the cytochrome P450 system to their corresponding oxon analogues to act as potent inhibitors of acetylcholinesterase. It is known that interactions between cytochrome P450 and pesticides include the inhibition of major xenobiotic metabolizing cytochrome P450 enzymes and changes on the genetic level.
METHODS
In this study, the influence of the pesticides parathion and paraoxon on human cytochrome P450 and associated oxygenases was investigated with a metabolically competent cell line (HepaRG cells). First, the viability of the cells after exposure to parathion and paraoxon was evaluated. The inhibitory effect of both pesticides on cytochrome P450 3A4, which is a pivotal enzyme in the metabolism of xenobiotics, was examined by determining the dose-response curve. Changes on the transcription level of 92 oxygenase associated genes, including those for important cytochrome P450 enzymes, were evaluated.
RESULTS
The exposure of HepaRG cells to parathion and paraoxon at concentrations up to 100 µM resulted in a viability of 100 per cent. After exposure for 24 hours, pronounced inhibition of cytochrome P450 3A4 enzyme activity was shown, indicating 50 per cent effective concentrations of 1.2 µM (parathion) and 2.1 µM (paraoxon). The results revealed that cytochrome P450 involved in parathion metabolism were significantly upregulated.
DISCUSSION
Relevant changes of the cytochrome P450 3A4 enzyme activity and significant alteration of genes associated with cytochrome P450 suggest an interference of pesticide exposure with numerous metabolic processes. The major limitations of the work involve the use of a single pesticide and the model as surrogate to human hepatocytes.
CONCLUSION
The data of this study might be of relevance after survival of acute, life-threatening intoxications with organophosphorus compounds, particularly for the co-administration of drugs, which are metabolized by the affected cytochrome P450.
Topics: Humans; Paraoxon; Parathion; Cell Survival; Pesticides; Dose-Response Relationship, Drug; Cytochrome P-450 Enzyme System; Cytochrome P-450 CYP3A; Insecticides; Cell Line; Cholinesterase Inhibitors
PubMed: 38874383
DOI: 10.1080/15563650.2024.2361879 -
Laboratory Animal Research Jun 2024The aim of the study was to develop a technique for quantitative determination of rat urine metabolites by HPLC-MS/MS, which can be used to search for biomarkers of...
BACKGROUND
The aim of the study was to develop a technique for quantitative determination of rat urine metabolites by HPLC-MS/MS, which can be used to search for biomarkers of acute intoxication with organophosphates (OPs).
RESULTS
The content of metabolites in the urine of rats exposed to a single dose of paraoxon (POX1x); interval, twice daily administration of paraoxon (POX2x); exposure to 2-(o-cresyl)-4H-1, 3, 2-benzodioxaphosphorin-2-oxide and paraoxon (CBPOX) was investigated. New data were obtained on the content in the urine of intact rats as well as rats in 3 models of OP poisoning: 3-methylhistidine, threonine, creatine, creatinine, lactic acid, acetylcarnitine, inosine, hypoxanthine, adenine, 3-hydroxymethyl-butyrate and 2-hydroxymethyl-butyrate.
CONCLUSIONS
The proposed assay procedure is a simple and reliable tool for urine metabolomic studies. Within 1-3 days after OP exposure in all three models of acute intoxication, the concentration of metabolites in rat urine, with the exception of adenine, changes similarly and symmetrically, regardless of the method of poisoning modeling, in all three models of acute intoxication. Further studies are needed to determine the specificity and reliability of using urinary metabolite concentration changes as potential biomarkers of acute organophosphate intoxication.
PubMed: 38845041
DOI: 10.1186/s42826-024-00209-3 -
Biomolecules May 2024Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood-brain barrier, and has demonstrated excellent safety and tolerability...
Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood-brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10-30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes.
Topics: Humans; Butyrylcholinesterase; Acetylcholinesterase; Ligands; Oximes; Cholinesterase Reactivators; Cholinesterase Inhibitors; Cholestenones; Kinetics; Sarin; GPI-Linked Proteins; Antidotes; Cholesterol; Organophosphorus Compounds
PubMed: 38785995
DOI: 10.3390/biom14050588 -
International Journal of Biological... Jun 2024In this present study, characteristics and structure-function relationship of an organophosphate-degrading enzyme from Bacillus sp. S3wahi were described. S3wahi...
Identification and characterization of a promiscuous metallohydrolase in metallo-β-lactamase superfamily from a locally isolated organophosphate-degrading Bacillus sp. strain S3wahi.
In this present study, characteristics and structure-function relationship of an organophosphate-degrading enzyme from Bacillus sp. S3wahi were described. S3wahi metallohydrolase, designated as S3wahi-MH (probable metallohydrolase YqjP), featured the conserved αβ/βα metallo-β-lactamase-fold (MBL-fold) domain and a zinc bimetal at its catalytic site. The metal binding site of S3wahi-MH also preserves the H-X-H-X-D-H motif, consisting of specific amino acids at Zn1 (Asp69, His70, Asp182, and His230) and Zn2 (His65, His67, and His137). The multifunctionality of S3wahi-MH was demonstrated through a steady-state kinetic study, revealing its highest binding affinity (K) and catalytic efficiency (k/K) for OP compound, paraoxon, with values of 8.09 × 10 M and 4.94 × 10 M s, respectively. Using OP compound, paraoxon, as S3wahi-MH native substrate, S3wahi-MH exhibited remarkable stability over a broad temperature range, 20 °C - 60 °C and a broad pH tolerance, pH 6-10. Corresponded to S3wahi-MH thermal stability characterization, the estimated melting temperature (T) was found to be 72.12 °C. S3wahi-MH was also characterized with optimum catalytic activity at 30 °C and pH 8. Additionally, the activity of purified S3wahi-MH was greatly enhanced in the presence of 1 mM and 5 mM of manganese (Mn), showing relative activities of 1323.68 % and 2073.68 %, respectively. The activity of S3wahi-MH was also enhanced in the presence of DMSO and DMF, showing relative activities of 270.37 % and 307.41 %, respectively. The purified S3wahi-MH retained >60 % residual activity after exposure to non-ionic Tween series surfactants. Nevertheless, the catalytic activity of S3wahi-MH was severely impacted by the treatment of SDS, even at low concentrations. Considering its enzymatic properties and promiscuity, S3wahi-MH emerges as a promising candidate as a bioremediation tool in wide industrial applications, including agriculture industry.
Topics: Bacillus; beta-Lactamases; Kinetics; Substrate Specificity; Enzyme Stability; Hydrogen-Ion Concentration; Catalytic Domain; Amino Acid Sequence; Organophosphates; Bacterial Proteins; Temperature
PubMed: 38761915
DOI: 10.1016/j.ijbiomac.2024.132395 -
International Journal of Systematic and... May 2024A bacterial strain designated MMS21-TAE1-1, capable of degrading paraoxon, was isolated from red pepper soil (36° 25' 26.0″ N, 126° 25' 47.0″ E) and subjected to...
A bacterial strain designated MMS21-TAE1-1, capable of degrading paraoxon, was isolated from red pepper soil (36° 25' 26.0″ N, 126° 25' 47.0″ E) and subjected to polyphasic taxonomic characterisation. MMS21-TAE1-1 was an aerobic, non-motile and Gram-stain-positive bacterium. MMS21-TAE1-1 showed growth at 10-37 °C (optimum, 30 °C), at pH 4-10 (optimum, pH 7) and in the presence of 0-6 % NaCl (optimum, 0 %). On the basis of the results of 16S rRNA gene sequence analysis, MMS21-TAE1-1 could be assigned to the genus and shared the highest sequence similarities with NBRC 12136 (99.72 %), then with G2-1 (99.65 %) and DSM 20138 (99.17 %). However, the results of genome-based comparison using orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridisation indicated that MMS21-TAE1-1 could be readily distinguished from all species of the genus with validly published names. The predominant menaquinone of MMS21-TAE1-1 was MK-9(H). The diagnostic polar lipids were diphosphatidylglycerol and phosphatidylinositol, and unidentified glycolipids were also present. The major fatty acids were anteiso-C, anteiso-C, iso-C and iso-C. The chemotaxonomic properties of MMS21-TAE1-1 were generally consistent with those of members of the genus . The genome of MMS21-TAE1-1 contained genes related to degradation of aromatic compounds. It is evident from the results of this study that strain MMS21-TAE1-1 merits recognition as representing a novel species of the genus , for which the name sp. nov. is proposed. The type strain is MMS21-TAE1-1 (=KCTC 49652 = LMG 32368).
Topics: Soil Microbiology; RNA, Ribosomal, 16S; Fatty Acids; Phylogeny; DNA, Bacterial; Sequence Analysis, DNA; Bacterial Typing Techniques; Nucleic Acid Hybridization; Vitamin K 2; Base Composition; Capsicum
PubMed: 38752999
DOI: 10.1099/ijsem.0.006389 -
Inorganic Chemistry May 2024The ability to control the catalytic activity of enzymes in chemical transformations is essential for the design and development of artificial catalysts. Herein, we...
The ability to control the catalytic activity of enzymes in chemical transformations is essential for the design and development of artificial catalysts. Herein, we report the synthesis and characterization of functional ligands featuring two 1,4,7,10-tetraazacyclododecane units linked by an azobenzene group and their corresponding dinuclear Zn(II) complexes. We show that the configuration switching () of the azobenzene spacer in the ligands and their dinuclear Zn(II) complexes is reversibly controlled by irradiation with UV and visible light. The Zn(II)-metal complexes are light-responsive catalysts for the hydrolytic cleavage of nerve agent simulants, i.e., -nitrophenyl diphenyl phosphate and methyl paraoxon. The catalytic activity of the -isomers of the dinuclear Zn(II) complexes outperformed that of the -counterparts. Moreover, combining the less active -isomers with gold nanoparticles induced an enhancement in the hydrolysis rate of -nitrophenyl diphenyl phosphate. Kinetic analysis has shown that the catalytic site appears to involve a single metal ion. We explain our results by considering the different desolvation effects occurring in the catalyst's configurations in the solution and the catalytic systems involving gold nanoparticles.
PubMed: 38747890
DOI: 10.1021/acs.inorgchem.4c01061 -
Toxicology Mechanisms and Methods May 2024Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the...
Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 µM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from the control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms, including inflammatory response, oxidative stress or activation of apoptosis to further understand the nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme-inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.
PubMed: 38745427
DOI: 10.1080/15376516.2024.2356184 -
Brain Injury May 2024Organophosphorus pesticide poisoning can lead to severe brain damage, but the specific mechanisms involved are not fully understood. Our research aims to elucidate the...
BACKGROUND
Organophosphorus pesticide poisoning can lead to severe brain damage, but the specific mechanisms involved are not fully understood. Our research aims to elucidate the function of the TRPV4 ion channel in the development of brain injury induced by paraoxon (POX).
METHODS
, we examined the survival rate, behavioral seizures, histopathological alterations, NMDA receptor phosphorylation, as well as the expression of the NLRP3-ASC-caspase-1 complex and downstream inflammatory factors in the POX poisoning model following intervention with the TRPV4 antagonist GSK2193874. , we investigated the effects of GSK2193874 on NMDA-induced inward current, cell viability, cell death rate, and Ca accumulation in primary hippocampal neurons.
RESULTS
The treatment with the TRPV4 antagonist increased the survival rate, suppressed the status epilepticus, improved pathological damage, and reduced the phosphorylation level of NMDA receptors after POX exposure. Additionally, it inhibited the upregulation of NLRP3 inflammasome and inflammatory cytokines expression after POX exposure. Moreover, the TRPV4 antagonist corrected the NMDA-induced increase in inward current and cell death rate, decrease in cell viability, and Ca accumulation.
CONCLUSION
TRPV4 participates in the mechanisms of brain injury induced by POX exposure through NMDA-mediated excitotoxicity and NLRP3-mediated inflammatory response.
PubMed: 38711413
DOI: 10.1080/02699052.2024.2351104