Johnson Martin KeithConsultant Toxicologist, c/o MRC Toxicology Unit, Hodgkin Building, University of Leicester, Lancaster Road, P.O. Box 138, Leicester LEI 9HN United KingdomPhone : Fax :
Dr. Martin Keith Johnson, born in 1930, died in Banstead, Surrey on the 9th November 2018. Dr. Johnson was a founder member of the British Toxicology Society.In the early years of his carrier he worked in the Biochemical Mechanisms Group run by Dr Norman Aldridge. He discovered the neuropathy target esterase (NTE) as an enzyme activity, inhibited by low concentrations of the neuropathic OP, mipafox, but resistant to an AChE-directed OP, paraoxon. A paper by Ted Lock, Andrew Smith and Paul Glynn retraces his achievements Obituary
Title: The Potential Value of beta-Amyloid Imaging for the Diagnosis and Management of Dementia: A Survey of Clinicians Zhong Y, Karlawish J, Johnson MK, Neumann PJ, Cohen JT Ref: Alzheimer Disease & Associated Disorders, 31:27, 2017 : PubMed
We assessed the potential influence of beta-amyloid imaging on clinician diagnoses and management of patients with memory loss. We surveyed 315 clinicians, assigning each a vignette describing a hypothetical patient with symptoms of unexplained mild cognitive impairment, possible Alzheimer disease (AD), or young-onset dementia. Vignettes reported "positive," "negative," or no beta-amyloid imaging information. We assessed imaging's influence on diagnosis (AD contribution to symptoms), diagnostic confidence, and patient management. Compared with clinicians receiving no imaging, clinicians given positive imaging results more often attributed symptoms to AD [odds ratio (OR)=5.91; 95% confidence interval (CI), 1.25-27.97]; clinicians given negative imaging were less likely (OR=0.10; 95% CI, 0.04-0.21). Clinicians identifying AD as contributing to symptoms more often recommended acetylcholinesterase inhibitor (AChEI) (OR=18.59; 95% CI, 6.86-50.36) and N-methyl-D-aspartate receptor antagonists (OR=3.63; 95% CI, 1.78-7.39). We found that negative imaging reduced AChEI recommendations. Positive imaging reduced recommendation of beta-amyloid imaging for future patients. In conclusion, beta-amyloid imaging can influence diagnosis, prescriptions, and patient management.
Title: Toxicological assessment of isomeric pesticides: a strategy for testing of chiral organophosphorus (OP) compounds for delayed polyneuropathy in a regulatory setting Battershill JM, Edwards PM, Johnson MK Ref: Food & Chemical Toxicology, 42:1279, 2004 : PubMed
Many compounds, including some pesticides, contain structural centres of asymmetry, which convey the property of a type of stereoisomerism known as chirality. Such compounds can exist in two or more forms, depending on the number of chiral atoms and are termed stereoisomers or enantiomers. Stereoisomers of a particular compound can have different biological properties; one such of particular importance for toxicological evaluation, is the potential for differences in metabolic disposal of and binding of stereoisomers to molecular targets in the cell. The combination of differential metabolism of chiral organophosphorus (OP) pesticides and opposing stereoselectivity of inhibition of neuropathy target esterase (NTE) and acetylcholinesterase (AChE) can affect the value of the hen test, performed to OECD guidelines, in predicting the potential to cause organophosphate-induced delayed polyneuropathy (OPIDP) in humans. This is a mixed central and sensory and motor neuropathy. The experimental data on structural analogues of the pesticide methamidophos and the evidence for stereoselective OPIDP are reviewed and a model is given demonstrating how the properties of a chiral OP can result in the neuropathic potential not being detected by the standard hen test. A strategy for the assessment of a racemic mixture comprised of two OP enantiomers for the potential to induce OPIDP is outlined. The strategy uses information from structure activity relationships (SAR), in vitro tests and in vivo tests to allow risk assessment decisions to be made. It is suggested that the potential for stereoselective toxicity of pesticides should be routinely considered in regulatory assessments.
Title: W. Norman Aldridge Johnson MK Ref: Toxicol Sci, 59:3, 2001 : PubMed
Paper
Norman Aldridge was fascinated by the interaction of chemicals with living systems. He firmly believed that understanding how and why chemicals exert their toxic effects was fundamental to safety evaluation for chemicals used in the workplace or environment. He loved research with 'an insatiable urge to make sense of things,' but also loved to apply the results to real-life issues. He encouraged his students, postdoctoral fellows, and visiting scientists from around the world to do the same.
From the age of 16, Norman worked as technician in a dye works. His interest in toxicology started during World War II at the U.K. Chemical Defence Establishment at Porton, where he worked as an assistant to the renowned physiologist Professor Sir Charles Lovatt-Evans on practical problems of protection against chemical warfare agents. He proved himself a careful investigator, and in part-time study, he obtained a B.Sc. in chemistry and physiology. Norman cited Lovatt-Evan's dictum, 'I don't think it will work, but you try it,' as a formative influence on his attitude, and subsequently it rubbed off onto many of his own students and colleagues. In 1947 John Barnes, who was also at Porton at that time, was appointed director of the Medical Research Council's new Toxicology Unit located at Carshalton, Surrey. Barnes' remit was broad: 'Do something' to help face health problems that might arise as the postwar chemical industry burgeoned. He recruited Norman as the unit's first scientific staff member. Over the next 40 years, Norman worked on the mechanism of toxicity of chemicals, including beryllium, organophosphorous compounds, acrylamide, organotin and organolead compounds, and pyrethroid insecticides. He drew visitors from all over the world but never built an 'empire' -preferring to train, encourage, and liberate his students.
In his early work, Aldridge established much of the basic enzymology on the interaction of organophosphorus esters with esterases. He classified these into A, B, or C esterases according to whether organophosphorus esters were hydrolyzed by them, were inhibited, or did not interact either way. With Elsa Reiner, Aldridge worked on the interaction of carbamate esters with acetylcholinesterase. Together they wrote a textbook entitled Interaction of Esterases with Esters of Organophosphorous and Carbamic Acids (Elsevier, North Holland, 1972), which is still a standard text. Aldridge initiated the search for the esterase that was thought to be associated with peripheral neuropathy caused by certain organophosphorus esters. He supported and encouraged Martin Johnson in his studies, which led to the discovery of what is now called 'neuropathy target esterase'.
In the 1950s he turned his attention to the toxicity of organotin and organolead compounds, the stimulus being an unfortunate poisoning that occurred in France when more than 100 people were treated for boils with a poultice containing triethyltin. From this event research developed over many years, which led to a wider understanding of the interaction of di- and trisubstituted tin and lead compounds with mitochondria and the process of oxidative phosphorylation. These chemicals are selectively toxic to the central nervous system and in collaboration with Alwyn Brown, Aldridge was the first to show that trimethyltin produces selective neuronal cell death in the hippocampus, in contrast to triethyltin, which produces edema in the myelin sheath.
Selective biological responses such as the above provided the stimulus for many of Aldridge`s investigations, and his scientific attitude is captured in his book entitled Mechanisms and Concepts in Toxicology (Taylor and Francis, 1996).
Throughout his career Norman persisted in laboratory work and revelled in the data generated by his students, visiting scientists, and colleagues, especially the anomalous findings that did not fit the current theory. He did not tolerate loose thinking and always had probing questions and suggestions on how to further test the data to lead to a clearer understanding of the findings. His terse questions 'Why?' and 'How?' jolted many a woolly thinker into more self-critical mode!
In 1966 he was appointed head of the Biochemical Mechanisms Section in the Medical Research Council Toxicology Unit, a post he held until he retired in 1985; he was also deputy director for his last 10 years. Following his retirement he took up a position as Professor of Biochemical Toxicology at the University of Surrey, where he actively contributed to the teaching of toxicology to postgraduate students. He received many awards. He was the founder Chairman of British Toxicology Society, first recipient of the John Barnes award of the Society, and was the first honorary member of the Society. Both the European Societies of Toxicology (EUROTOX) and the American Society of Toxicology bestowed upon him their Merit Award in recognition of his contribution to the science of toxicology. In 1977 Queen Elizabeth II awarded him the Order of the British Empire for his services to toxicology. He was active internationally in toxicology as a member and then as secretary-general of the International Union of Toxicology. Norman was keen to promote European collaboration and the exchange of students in toxicology. As a member and then chairman of the European Science Foundation programme of Grants in Toxicology, he was able to stimulate such interactions.
During his career Aldridge's advice was sought both nationally and internationally on issues related to toxicology. His interest in tackling real issues meant he was an advisor in many of the major chemical disasters over the last four decades, such as the contamination of cooking oils in Morocco with tri-ortho-cresyl phosphate, the toxic oil epidemic in Spain, and the pesticide plant explosion in Bhopal, India. He played an active role as the Director of the World Health Organization collaborating laboratory, based at Carshalton, in the poisoning incident with malathion, when several thousand malaria control sprayers in Pakistan became ill and five died. He conducted a series of studies which showed that malathion upon storage under hot and humid conditions degraded to form isomalathion. He further showed that this impurity was a good inhibitor of carboxylesterase enzymes in the liver, which in healthy persons readily degrades any malathion that may enter the body during normal working conditions. This basic work led to changes in the manufacturing and storage procedures for malathion and other pesticides to prevent this type of event occurring again. Until shortly before his death in June 1996 after a brief illness, he was advising on the problem of postevent identification of signs in a patient of a transient neurotoxin the notorious 'Case of the Poisoned Professor' in New Zealand; he successfully advocated use of a mass spectrometric biomonitoring method of detection of exposure to acrylamide developed by colleagues in the Toxicology Unit. He fertilized the field of toxicology worldwide with a passion for understanding molecular mechanisms that will never be erased from the discipline. It is fitting that the British Toxicology Society has recently established Norman Aldridge Travelling Fellowships to support international visits enabling young scientists to widen their experience of research in toxicology.
Title: Identification of acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds and a potential target for cognitive enhancing drugs Richards PG, Johnson MK, Ray DE Ref: Molecular Pharmacology, 58:577, 2000 : PubMed
We describe here the purification and identification of a previously unrecognized target for organophosphorus compounds. The target, acylpeptide hydrolase, was isolated as a tritiated-diisopropylfluorophosphate-reactive protein from porcine brain and purified to homogeneity using a combination of ion-exchange and gel-filtration chromatography. Biochemical characterization and internal sequence analysis confirmed identity. Acylpeptide hydrolase was found to be potently inhibited by the organophosphorus compounds chlorpyrifosmethyl oxon, dichlorvos, and diisopropylfluorophosphate (20-min IC(50) values of 18.3 +/- 2.0, 118.7 +/- 9.7, and 22.5 +/- 1.2 nM, respectively). The in vitro sensitivity of acylpeptide hydrolase toward these compounds is between six and ten times greater than that of acetylcholinesterase (AChE), making it a target of pharmacological and toxicological significance. We show that, in vivo, acylpeptide hydrolase is significantly more sensitive than AChE to inhibition by dichlorvos and that the inhibition is more prolonged after a single dose of inhibitor. Furthermore, using dichlorvos as a progressive inhibitor, it was possible to show that acylpeptide hydrolase is the only enzyme in the brain capable of hydrolyzing the substrate N-acetyl-alanyl-p-nitroanilide. A concentration of 154 +/- 27 pmol of acylpeptide hydrolase/gram of fresh rat brain was also deduced by specific labeling with tritiated-diisopropylfluorophosphate. We also suggest that, by comparison of structure-activity relationships, acylpeptide hydrolase may be the target for the cognitive-enhancing effects of certain organophosphorus compounds. Acylpeptide hydrolase cleaves N(alpha)-acylated amino acids from small peptides and may be involved in regulation of neuropeptide turnover, which provides a new and plausible mechanism for its proposed cognitive enhancement effect.
Title: Neuropathy target esterase (NTE) and organophosphorus-induced delayed polyneuropathy (OPIDP): recent advances Johnson MK, Glynn P Ref: Toxicol Lett, 82-83:459, 1995 : PubMed
The identification of neuropathy target esterase (NTE) as the site for initiation of organophosphorus-induced delayed polyneuropathy (OPIDP) has led to informative acute and chronic neurotoxicity tests (adopted by OECD and EPA), to structure/activity and in vitro/in vivo predictions, and to a sound basis for extrapolations to man. Purification of the sodium dodecyl sulphate (SDS)-denatured 155-kDa sub-unit of NTE has enabled partial sequencing and molecular biological studies. A MAb to the chicken brain sub-unit and PAbs to synthetic peptides have been raised: preliminary experiments suggest that one is effective for immunohistochemistry of frozen tissue. cDNA libraries are being screened with synthetic oligonucleotides, polymerase chain reaction (PCR)-developed primers, and with Ab in order to obtain cloned NTE. Previous studies of NTE in vivo have not revealed its normal physiological function or the route from inhibition to degeneration of axons, but the current progress in molecular biology of NTE is applicable to study of the function of normal and organophosphorus (OP)-modified NTE in cultured neural cells.
In 1990 an outbreak of ataxia occurred in over 700 pigs in the north of England. Epidemiological studies demonstrated that the disorder was associated with the consumption of feed from a particular supplier and that one component (wheat screenings) was common to the batch of feed with which the ataxia was associated. An analysis of the feed demonstrated the presence of an organophosphorus pesticide, later identified as isofenphos, a pesticide not approved for use in the United Kingdom. The wheat screenings had been imported from France and the warehouse in which they had been stored was contaminated with isofenphos, which is approved for restricted use in France. Isofenphos is known to cause delayed neuropathy. The dose to which the pigs were theoretically exposed would be expected to have resulted in neuropathy (manifested as ataxia).
Title: Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase Glynn P, Read DJ, Guo R, Wylie S, Johnson MK Ref: Biochemical Journal, 301 ( Pt 2):551, 1994 : PubMed
We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.
Title: Molecular characterisation of neuropathy target esterase: proteolysis of the [3H]DFP-labelled polypeptide Glynn P, Ruffer-Turner M, Read D, Wylie S, Johnson MK Ref: Chemico-Biological Interactions, 87:361, 1993 : PubMed
Neuropathy target esterase (NTE) in hen brain membranes can be labelled with tritiated di-isopropylfluorophosphate ([3H]DFP) and appears to be associated with a 155-kDa polypeptide. Using preparative SDS-PAGE, we have obtained preparations in which [3H]DFP-labelled NTE comprises 2% of the total protein. Further purification of the 155-kDa polypeptide has proved difficult. We therefore attempted to use proteases to excise smaller [3H]DFP-labelled fragments which might be more amenable to fractionation. V8 protease treatment generated a labelled fragment of about 16 kDa which could be fractionated on SDS-PAGE and contained tritium attached to both site X (putatively the active site serine) and site Z (the residue to which an isopropyl moiety is transferred during aging of [3H]DFP-inhibited NTE). Papain and thermolysin treatments generated a small labelled peptide (< 10 kDa) which could be fractionated on reverse-phase HPLC and in which tritium was attached to site X but not site Z. N-terminal sequencing of the thermolysin-generated peptide fraction indicated sample heterogeneity but also suggested that the active site of NTE may contain the serine esterase consensus sequence: Gly-Glu-Ser-Xxx-Gly.
Title: Symposium introduction: retrospect and prospects for neuropathy target esterase (NTE) and the delayed polyneuropathy (OPIDP) induced by some organophosphorus esters Johnson MK Ref: Chemico-Biological Interactions, 87:339, 1993 : PubMed
This article introduces a Symposium devoted to Neuropathy Target Esterase (NTE). The characteristics of the disorder known as OPIDP are described and the steps by which NTE was identified as the target are summarised. Studies with many organophosphates, phosphinates and chiral phosphonates are entirely consistent with a 2-step process of initiation referred to as 'NTE (70-80%) aging': about 70-80% of available nervous system NTE is first covalently phosphylated causing inhibition of esterase activity, and then the molecules of inhibited NTE undergo a covalent bond-cleavage leaving a negative charge in the region of the still-bound phosphorus. This understanding has clarified structure/activity studies of neuropathic potential of OP esters and is now routinely applied in toxicological evaluations for regulatory purposes. However, the biological function of NTE has remained a mystery. Prospective views of the role of NTE are presented by different authors. Attempts to isolate catalytically active or radiolabelled inhibited NTE are near to success. Since the Symposium, complete isolation of NTE affinity-purified from hen brain has been reported (see M.K. Johnson & P. Glynn, Toxicologist, 13 (1993) 211, Abstr. 773). Some minor, but possibly significant, differences in properties of a soluble and a membrane-bound form of NTE in sciatic nerve has been identified. The nature of the disturbance brought about by covalent binding of organophosphoramidates at the active site of NTE and the discovery that 'non-aging' inhibitors of NTE can promote neuropathy in hens given a sub-neuropathic dose of neuropathic OPs has led to a concept of NTE inhibitors having a range of 'partial agonist' effects at the covalent binding site. Evidence is emerging that the promotion target may be 'cousin-of NTE' with very similar inhibition characteristics and a function in the processes of response to or repair of axonal damage.
Title: Prophylaxis against and promotion of organophosphate-induced delayed neuropathy by phenyl di-n-pentylphosphinate Johnson MK, Read DJ Ref: Chemico-Biological Interactions, 87:449, 1993 : PubMed
Phenyl di-n-pentylphosphinate (PPP) is a potent inhibitor of neuropathy target esterase (NTE) with negligible effect on acetylcholinesterase: I50S at 37 degrees C for 20 min and pH 8, respectively are 0.2 microM and > 2mM. PPP is not neuropathic. This is compatible with the fact that inhibited NTE is autopsy material from hens dosed with PPP can always be reactivated in vitro, presumably because no 'aging' reaction has occurred. PPP (10 mg/kg s.c.) given to hens up to 4 days before severely neuropathic doses (1.7 mg/kg) of diisopropylphosphorofluoridate (DFP) prevented neuropathic but not cholinergic effects of DFP. Hens given PPP 3 days after a sub-neuropathic dose of DFP (0.4 mg/kg) developed severe clinical neuropathy (clinical scores of 7 and 5 compared with DFP-plus-solvent scores 0,1,3). These prophylactic and promoting effects are similar to those exerted by phenylmethanesulphonyl fluoride (PMSF) at doses which inhibit NTE. In 3 out of 4 birds a pre-dose with PMSF (15 mg/kg) prevented the promoting effect of 120 mg/kg PMSF given after DFP.
Title: Screening of O-ethyl O-4-nitrophenyl phosphoramidate (ENPP) for delayed neuropathic potential Johnson MK, Read DJ Ref: Chemico-Biological Interactions, 87:439, 1993 : PubMed
O-Ethyl-O-4-nitrophenylphosphoramidate is a short-acting anticholinesterase and a possible candidate for a prophylactic agent against nerve agents since human acetylcholinesterase inhibited by this agent undergoes rapid spontaneous reactivation which can be accelerated further, if necessary, by treatment with oximes. Doses of the agent > 1 mg/kg (s.c.) given to unprotected rats were fatal in a short time but 2 rats and one hen given 0.5 mg/kg survived. Hens given 2.5 or 4 mg/kg s.c. 20 min after prophylactic physostigmine + atropine survived acute effects and were killed 4.5 or 24 h later. Brain and spinal cord neuropathy target esterase levels of these hens were depressed only 4-10% compared with levels in brains from hens given only oxime + atropine or of undosed animals. Clinical signs of neuropathy were not seen in surviving birds observed for 3 weeks. It appears there would be negligible delayed neuropathic hazard associated with administration of O-ethyl-O-4-nitrophenylphosphoramidate at subacute doses.
Title: Stereo-specific degradation of the R-(+) isomer of O-n-hexyl-S-methylphosphorothioamidate catalysed by rabbit serum Johnson MK, Read DJ Ref: Chemico-Biological Interactions, 87:133, 1993 : PubMed
Resolved isomers of O-n-hexyl-S-methylphosphorothioamidate (HXM) which had been synthesised by separate stereospecific routes were analysed by chiral glc: about 2-3% of R-(+) isomer was found in the S-(-) sample and accounted for nearly all the inhibitory power against neuropathy target esterase. Incubation of racemic HXM with rabbit serum led to slow but very specific disposal of R-(+) isomer to undetectable levels with very slight loss of S-(-): the rate of disposal was roughly estimated to be about 1% of the published rate of hydrolysis of paraoxon. Incubation with crystalline chymotrypsin caused a preferential but not totally selective disposal of S-(-) isomer.
Title: The R-(+)isomer of O-n-hexyl S-methyl phosphorothioamidate causes delayed neuropathy in hens after generation of a form of inhibited neuropathy target esterase (NTE) which can be reactivated ex vivo Johnson MK, Safi JM Ref: Chemico-Biological Interactions, 87:443, 1993 : PubMed
To initiate delayed neuropathy (DN) in adult hens organophosphates and phosphonates must inhibit most neural NTE and the inhibited NTE must undergo an 'aging' reaction. Phosphinates and those chiral isomers of phosphonates which produce non-aging NTE do not cause DN but act as prophylactic agents. Some racemic phosphoramidates cause DN although the inhibited NTE in autopsy samples can be reactivated in vitro (Johnson, Read and Vilanova, 1991, Arch. Toxicol., 65, 618-624). We now report that pure R(+)isomer of O-n-hexyl S-methyl phosphorothioamidate (5-20 mg/kg per os) caused slight acute effects but typical DN associated with high inhibition of NTE in brain, spinal cord and sciatic nerve (maximum by 6-24 h): the inhibited NTE was easily reactivated by KF (presumed not aged). For each dose the average residual NTE activity in the three tissues 24 h after dosing and the clinical ataxia severity on peak days 15-17 (score out of 4) was: 5 mg/kg: 13, 14, 27% (2,2,2,1); 10 mg/kg: 10, 14, 12%, (4,3,2); 15 mg/kg: 10,11,17%, (3,3,4); 20 mg/kg: 6, 10, 8% (3,3,3,2). The ability of this isomer and of other racemic phosphoramidates to initiate DN by covalent reaction at the active site of NTE (inhibition) without subsequent aging suggests that the chemistry (? charge distribution) in the region of the phosphorus atom determines that disturbance in the molecular environment of NTE which initiates DN.
Title: Interactions in vitro of some organophosphoramidates with neuropathy target esterase and acetylcholinesterase of hen brain Jokanovic M, Johnson MK Ref: Journal of Biochemical Toxicology, 8:19, 1993 : PubMed
For organophosphates or phosphonates to initiate delayed neuropathy two steps are necessary: (1) progressive covalent reaction with neuropathy target esterase (NTE) to produce a form of inhibited NTE which can be reactivated by incubation with aqueous potassium fluoride (KF) and (2) progressive "aging" of inhibited NTE to a form which can no longer be reactivated by KF. However, it has been shown recently that certain N-unsubstituted organophosphoro-monoamidates (analogues of methamidophos) cause delayed neuropathy even though the inhibited NTE appeared not to have aged (Johnson et al. (1991). Arch. Toxicol., 65, 618-624). In order to study the generality of this phenomenon, we have examined some N-substituted compounds. We report in vitro studies of inhibition and reactivation and aging of both NTE and acetylcholinesterase (AChE) prior to toxicological tests. All the compounds studied were less inhibitory to both NTE and AChE in concentrated rather than in dilute suspensions of EDTA-washed brain particles without added cofactors. There was an apparent disposal of up to 100 mumoles of test compound by particles from 95 mg hen brain, which is far greater than can be explained by covalent binding. The activity is distinct from calcium-dependent "A" esterase. Several N-alkyl phosphoromonoamidates were found to be potent and selective inhibitors of NTE: second-order rate constant for O-n-pentyl N-benzylphosphoramido-fluoridate (Cmpd 6) = 5.6 x 10(7) M-1 min-1 at 37 degrees, which is about 100x higher than for acetylcholinesterase (AChE). Inhibited NTE and AChE from several chiral phosphoromono-amidates did not reactivate spontaneously (21 hours at 37 degrees). Virtually 100% reactivation by KF of AChE inhibited by phosphoromonoamidates was achieved at all times tested. Acetylcholinesterase inhibited by 2,5-dichlorophenyl N,N'-di-n-butylphosphorodiamidate was 42-56% reactivated by incubation with KF (192 mM in pH 5.2 buffer for 30 minutes at 37 degrees). We believe this is the first report of reactivation of any enzyme after inhibition by a phosphorodiamidate. For NTE inhibited by tabun (O-ethyl N-dimethylphosphoroamidocyanidate), virtually complete and rapid aging (t1/2 = 5.5-8.4 minutes) was observed. Consistent but only partial reactivation by KF was achieved 2 or more hours after inhibition of NTE by Cmpd 6 or by its 2,6-difluoro-analogue (Cmpd 7). However, a small but significant aging (approximately 15-20% loss of reactivatability) was measured soon after a 1 minute inhibition by Cmpd 7, but no further change occurred in 21 hours.
Title: Reactivation of phosphorodiamidated acetylcholinesterase and neuropathy target esterase by treatment of inhibited enzyme with potassium fluoride Milatovic D, Johnson MK Ref: Chemico-Biological Interactions, 87:425, 1993 : PubMed
It has been thought that the phosphorus-enzyme bond in inhibited esterases inhibited by such agents as mipafox (N,N'-di-iso-propylphosphorodiamidate) was refractory to reactivating agents either because an 'aging' reaction occurs soon after inhibition or because the bond was intrinsically very strong. We have found that both acetylcholinesterase (AChE) and neuropathy target esterase (NTE) which had been inhibited with either mipafox or with a di-n-butylphosphorodiamidate could be reactivated by prolonged treatment with aqueous potassium fluoride (KF): the reaction proceeded with first-order kinetics. Furthermore there was no time-dependent loss of reactivatability (aging). Di-isopropylphosphoro-butyrylcholinesterase could be fully reactivated by this treatment but after 18 h to allow aging the monoisopropyl phosphoro-enzyme was totally refractory to KF. We conclude that it is likely that the mipafox-enzyme bond in inhibited NTE and AChE is relatively strong but that aging has not occurred. The local disturbance around the active site of NTE caused by attachment of the phosphorodiamidate molecule appears to be sufficient to initiate delayed neuropathy without necessity for an 'aging' reaction.
Title: Hydrolysis of some organophosphorus dichlorophenyl esters by hen brain homogenates and rabbit serum compared with hydrolysis of paraoxon Reiner E, Johnson MK, Jokanovic M Ref: Chemico-Biological Interactions, 87:127, 1993 : PubMed
The hydrolysis of four organophosphorus dichlorophenyl esters and of paraoxon was studied in hen brain homogenates and in rabbit serum. All compounds were hydrolysed by both preparations, but the rates were different in the two preparations. EDTA inhibited the hydrolysis almost completely in rabbit serum, but had only a small effect on the hydrolysis in hen brain homogenates.
Title: Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved isomers and of some higher O-alkyl homologues Johnson MK, Vilanova E, Read DJ Ref: Archives of Toxicology, 65:618, 1991 : PubMed
The interaction with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) in vivo of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved stereoisomers and five higher O-alkyl homologues has been examined along with the ability of these compounds to cause organophosphorus-induced delayed polyneuropathy (OPIDP) in adult hens. For the lower homologues AChE was more sensitive than NTE and it was impossible to achieve high inhibition of NTE in vivo without both prophylaxis and therapy against acute anticholinesterase effects; for the n-hexyl homologue high inhibition of NTE could be achieved without obvious anticholinesterase effects and spontaneous reactivation of inhibited AChE was seen as in vitro. The maximum tolerated dose of L(-) methamidophos or of the ethyl or iso-propyl homologues did not inhibit NTE more than 60%, and surviving birds did not develop OPIDP. The n-propyl, n-butyl and n-hexyl compounds caused typical OPIDP at doses causing a peak of 70-95% inhibition of NTE in brain, spinal cord and sciatic nerve soon after dosing. Racemic methamidophos caused unusually mild OPIDP associated with very high inhibition of NTE at doses estimated to be greater than 8 times the unprotected LD50 and the D-(+) isomer caused OPIDP at about 5-7 x LD50. Clinical effects correlated with histopathology in 19 out of 20 examined birds. In contrast to results of many previous studies with organophosphates and phosphonates, all these cases of OPIDP were associated with formation of inhibited NTE which could be reactivated ex vivo by treatment of autopsy tissue with KF solution.
Title: Delayed neuropathy and acute toxicity studies with pirimiphos-methyl in the hen Lock EA, Johnson MK Ref: Journal of Applied Toxicology, 10:17, 1990 : PubMed
This paper describes studies aimed at determining the acute anticholinergic and delayed neurotoxic potential of the organophosphate insecticide pirimiphos-methyl (O-2-diethylamino-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate) in the hen. Delayed neuropathy was assessed by biochemical measurement of neuropathy target esterase (NTE) activities in the brain and spinal cord, clinical signs of neuropathy over two 21-day periods and histological assessment of nervous tissue. Acetylcholinesterase (AChE) activity was also determined in the brain and spinal cord. Hens were given a single oral dose of 100 mg kg-1 pirimiphos-methyl, which was followed by a repeated dose after 21 days. Tri-o-cresyl phosphate (TOCP), 500 mg kg-1, was used as a positive control. All pirimiphos-methyl-treated hens received prophylactic doses of N-methylpyridinium-2-aldoxime methanesulphonate (P2S) and atropine sulphate. Hens dosed with pirimiphos-methyl had very low AChE activities (less than 20% of control) in both the brain and spinal cord, 24 and 48 h after dosing. In the TOCP-treated hens, the activities were about 90% of control. NTE activities in the brain and spinal cord of pirimiphos-methyl-treated hens were identical to those in the controls, while they were profoundly inhibited (greater than 80%) in the TOCP-treated hens. All hens dosed with pirimiphos-methyl showed the expected signs of AChE inhibition and, following recovery, usually by Day 5, no clinical signs of delayed neuropathy were seen. The TOCP-treated hens developed clinical signs of neuropathy
Title: Biochemical and clinical tests of the delayed neuropathic potential of some O-alkyl O-dichlorophenyl phosphoramidate analogues of methamidophos (O,S-dimethyl phosphorothioamidate) Johnson MK, Vilanova E, Read DJ Ref: Toxicology, 54:89, 1989 : PubMed
The interaction in vivo of four O-alkyl O-2,5-dichlorophenyl phosphoramidates with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) and their ability to cause delayed polyneuropathy in hens has been examined. Previous studies in vitro (Vilanova, Johnson & Vicedo, Pestic. Biochem. Physiol., 28 (1987) 224) had led to the prediction that these compounds would not be neuropathic but, rather, would be prophylactic agents against organophosphorus-induced delayed polyneuropathy. In vivo the effects of these esters on the enzymes differ in 2 respects from effects in vitro: (i) Relative sensitivity of the enzymes was different: thus greater than 50% of brain NTE remained 24 h after an oral dose of 15 mg/kg of the n-hexyl ester while only 10-30% of AChE remained although NTE was the more sensitive enzyme in vitro; (ii) In no case could the inhibited NTE or AChE in autopsy samples from birds dosed with any of the 4 esters be reactivated by treatment with potassium fluoride in vitro: the inhibited enzymes produced by incubation of tissue with the esters in vitro had been reactivatable. Prophylaxis, with therapy in some cases, was required to prevent acute anticholinesterase poisoning when doses were sufficient to cause high inhibition of neural NTE. Inhibition in brain was typically 5-10% more than in spinal cord and 10-15% more than in sciatic nerve. Unambiguous signs of polyneuropathy (Grade 3 or more on an 8-point scale) were not seen in birds observed up to 3 weeks after doses which caused less than 70% inhibition of NTE in brain and spinal cord or less than 60% inhibition in sciatic nerve of pair-dosed birds assayed 24 h after dosing. Doses of 300, 10, 100 and 65 mg/kg, respectively, of the methyl, ethyl, n-butyl and n-hexyl esters caused greater than 70% inhibition of NTE in all 3 neural tissues and neuropathy in the majority of observed birds. Analysis of consolidated dose/response data from 36 assayed and 51 observed birds showed that effects of Grade 3 or more were produced in about 90% of birds when inhibition of NTE was greater than 90% in brain, greater than 85% in spinal cord or greater than 75% in sciatic nerve.
Title: Degradation by rat tissues in vitro of organophosphorus esters which inhibit cholinesterase Pla A, Johnson MK Ref: Biochemical Pharmacology, 38:1527, 1989 : PubMed
Hydrolytic "A"-esterase activities of various tissues of rat (plasma, liver, kidney, brain and intestinal mucosa) against selected OP esters of diverse structure as potential substrates (paraoxon, di-n-propyl paraoxon, di-n-butyl paraoxon, chlorpyrifos oxon, di-(4-phenyl butyl) phosphorofluoridate and the chiral isomers of ethyl 4-nitrophenyl phenylphosphonate) were studied. We have developed a sensitive and widely applicable assay depending on measuring decline in residual inhibitory power of any chosen OP against horse serum cholinesterase: for seven compounds examined so far I50s against BCHE ranged from 0.07 to 70 nM, and it is easy to monitor loss of OP starting from an initial 25 microM concentration. Progressive destruction rates were always highest in liver and plasma with activity sometimes detectable in kidney, brain but not in intestinal mucosa, but the ratios of activity between tissues differed for different substrates. At 25 microM/37 degrees/pH 7.2 hydrolysis rates ranged from 8500 nmol/min/g liver for di-(4-phenylbutyl) phosphorofluoridate down to 0.8 nmol/min for the butyl analogue of paraoxon; the rate for L(-) isomer of EPN oxon (23 nmol/min/g liver) was greater than 2x that for the D(+) isomer and for paraoxon. From our data we conclude that several OP hydrolases exist whose identity may be further characterised by use of selective substrates
Title: Central-peripheral delayed neuropathy caused by diisopropyl phosphorofluoridate (DFP): segregation of peripheral nerve and spinal cord effects using biochemical, clinical, and morphological criteria Lotti M, Caroldi S, Moretto A, Johnson MK, Fish CJ, Gopinath C, Roberts NL Ref: Toxicol Appl Pharmacol, 88:87, 1987 : PubMed
Systemic injection of diisopropyl phosphorofluoridate (DFP; 1 mg/kg, sc) causes delayed neuropathy in hens. This effect is associated with a high level of organophosphorylation of neuropathy target esterase (NTE) followed by an intramolecular rearrangement called "aging." Phenylmethanesulfonyl fluoride (PMSF) also attacks the active center of NTE but "aging" cannot occur. This compound does not cause neuropathy and protects against a subsequent challenge systemic dose of DFP. Intraarterial injection of DFP (0.185 mg/kg) into only one leg of hens caused a high NTE inhibition (greater than 80%) in the sciatic nerve of the injected leg, but not in other parts of the nervous system (37% average). A unilateral neuropathy with typical histopathological lesions developed in the injected leg. PMSF (0.55 mg/kg) injected into each sciatic artery caused 47% inhibition of sciatic nerve NTE but only 17-22% inhibition of NTE elsewhere; it did not produce clinical or histopathological lesions. When these hens were challenged with DFP (1 mg/kg, sc), high inhibition of residual-free NTE (greater than 85%) occurred throughout the nervous system and clinical signs of a syndrome different from the classical delayed neuropathy developed: this spinal cord type of ataxia was associated with histopathological lesions in the spinal cord but not in peripheral nerve. PMSF (1 mg/kg) injected into only one sciatic artery caused selective protective inhibition of sciatic nerve NTE of that leg. After systemic challenge by DFP, clinical effects expressed were a combination of spinal cord ataxia plus unilateral peripheral neuropathy. The challenge dose of DFP (1 mg/kg, sc) was insufficient to produce clear histopathological lesions in unprotected peripheral nerves although spinal lesions were found in these hens. Thus clinical evaluation of the peripheral nervous system by means of walking tests and a simple test of "leg retraction" reflexes was more sensitive and specific in diagnosis of peripheral neuropathy than was the histopathology.
Title: Interaction of some unsubstituted phosphoramidate analogs of methamidophos (O,S-dimethyl phosphorothioamidate) with acetylcholinesterase and neuropathy target esterase of hen brain Vilanova E, Johnson MK, Vicedo JL Ref: Pesticide Biochemistry and Physiology, 28:224 , 1987 : PubMed
At 37 C and pH 7.4-8.0, five higher O-alkyl analogs of methamidophos and four O-alkyl O-2,5-dichlorophenyl phosphoramidates all were more potent progressive inhibitors of hen brain AChE and neuropathy target esterase (NTE) than was methamidophos itself. For AChE, ka increased from 7.2 x 102 to 1.0 x 10 5 M-1 min-1 between methyl and n-hexyl S-methyl esters and from 9.3 x 10 3 to 8.9 x 10 5 M-1 min-1 between ethyl and n-hexyl dichlorophenyl analogs. For NTE, the ranges were from 16 to 7.9 x 10 4 for S-methyl esters, and were 9.7 x 10 4 to 7.8 x 10 6 M-1 min-1 for dichlorophenyl. S-methyl esters were more active against AChE than against NTE and all the dichlorophenyl esters were more active against NTE than against AChE. Spontaneous reactivation of 75-100% activity without aging of AChE was found after 19 hr incubation at 37 C after inhibition by all nine straight-chain alkyl analogs. After inhibition by O-isopropyl S-methyl phosphorothioamidate, some spontaneous reactivation with complete aging of all remaining inhibited AChE occurred during 19 hr. No spontaneous reactivation or aging of inhibited NTE was detected. It was concluded that the molecular structures of the inhibited enzymes obtained from equivalent compounds in the two series of inhibitors were identical and that the leaving groups were, therefore, S-methyl and O-2,5-dichlorophenyl, respectively. Although hen brain NTE inhibited by methamidophos in vitro did not age, cases of delayed neuropathy in man have been reported and, presumably, require aging as well as inhibition of NTE. Possible explanations of this apparent discrepancy include (i) the fact that methamidophos consists of two chiral forms and that the form seen to be active in vitro may be disposed of preferentially in vivo, (ii) the possibility of activation in vivo to a different inhibitor, (iii) differences between conformation and ease of aging of inhibited NTE in vitro and in vivo, and (iv) species differences.
Title: The effect of steric factors on the interaction of some phenylphosphonates with acetylcholinesterase and neuropathy target esterase of hen brain Johnson MK, Read DJ, Yoshikawa H Ref: Pesticide Biochemistry and Physiology, 25:133, 1986 : PubMed
At 37 C and pH 8.0 the resolved isomers of S-(4-chlorobenzyl or 2,4-dichlorobenzyl) ethyl phenylphosphonothiolate are moderate inhibitors of AChE of hen brain (ka = 300-1400 M-1min-1) and weak inhibitors of neuropathy target esterase (NTE) (ka = 40-240 M-1min-1). The two (R)P(+) isomers are more active against NTE than are the (S)P(-) while the opposite is true for AChE. NTE inhibited by either (R)P(+) isomer underwent slow spontaneous reactivation (about 30% in 18 hr) but (S)P(-)-inhibited NTE did not reactivate. No spontaneous reactivation was detected for AChE inhibited by either steric form. AChE inhibited by (R)p(+) isomers could be reactivated by N-methylpyridinium-2-aldoxime methanesulfonate and slow aging was detected (23% in 18 hr). NTE inhibited by (R)p(+) isomers could be reactivated by w-isonitrosoacetophenone and no significant aging occurred in 18 hr at 37degC. Neither of the enzymes could be reactivated 60 min after commencement of inhibition with saturated solutions of the (S)p(-) isomers. NTE inhibited by racemic EPNO in a 1-min incubation formed a mixture of rapidly aging (t1/2 = 1min) and apparently non-aging inhibited NTE. Since EPNO should form the same ethyl phenylphosphonylated enzymes as the isomers under study we conclude that the oxime-resistance of inhibited NTE after inhibition by these (S)p(-)-isomers is due to rapid formation of aged enzyme during the (necessarily) long inhibition period. Substantial inhibition of NTE in vivo should be possible in hens protected against cholinergic effects of a dose of either (R)P(+) isomer. The toxicological consequence of such inhibition (? neuropathy or protection) should be investigated
Title: The aging reaction of inhibited neuropathy target esterase - fundamental studies and toxicological significance Johnson MK Ref: In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases, (Brzin M, Barnard EA, Sket D, Eds) De Gruyter:463, 1984 : PubMed
Title: Poster 73. Interaction of the resolved isomers of soman with chicken brain acetylcholinesterase and neuropathy target esterase Johnson MK, Read DJ, Benschop HP Ref: In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases, (Brzin M, Barnard EA, Sket D, Eds) De Gruyter:, 1984 : PubMed
Title: Interaction of some trialkyl phosphorothiolates with acetylcholinesterase. Characterization of inhibition, aging and reactivation Clothier B, Johnson MK, Reiner E Ref: Biochimica & Biophysica Acta, 660:306, 1981 : PubMed
The reaction of bovine erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) with a set of structurally related phosphorothiolates was studied in order to investigate the properties of the phosphorylated enzymes and to identify the leaving group. OOS- and OOS-trimethyl phosphorothiolates and their triethyl analogues inhibit acetylcholinesterase reversibly and by progressive inhibition, and the phosphorylated enzymes undergo both spontaneous reactivation and aging. For each compound the enzyme-inhibitor dissociation constant, and the rate constants for inhibition (ka), reactivation and aging have been derived. The OOS-compounds are more potent inhibitors than the OOS-compounds, and the derived inhibited enzymes reactivate and age faster. By comparing reactivation and aging rate constants with those obtained from phosphorylated enzymes of known structure it was concluded that the leaving group of during phosphorylation is the S-alkyl. SSS-trimethyl and -triethyl phosphorothiolates also form reversible complexes and inhibit the enzyme progressively. With these inhibitors the phosphorylated enzymes did not reactivate either spontaneously or in response to oximes under conditions successful for the other inhibitors. The ka values (37 degrees C, pH 7.4) range from 30 M-1 X min-1 (OOS-trimethyl phosphorothiolate) to 6.7 X 10(3) M-1 X min-1 (OOS-triethyl phosphorothiolate) as compared to 1.25 X 10(5) M-1 X min-1 determined for isomalathion (O, S-dimethyl S-(1,2-dicarbethoxyethyl)-phoshporodithioate), which was used as one of the reference compounds. If the inhibitory potency of the trialkyl phosphorothiolates is calculated from measurements made after a fixed preincubation time the results in ka values will be misleading.
Title: Initiation of organophosphate neurotoxicity [letter] Johnson MK Ref: Toxicol Appl Pharmacol, 61:480, 1981 : PubMed
Title: Delayed neurotoxicity caused by chronic feeding of organophosphates requires a high-point of inhibition of neurotoxic esterase Johnson MK, Lotti M Ref: Toxicol Lett, 5:99, 1980 : PubMed
Experiments are reported showing that chronic feeding of either mono-2-cresyl diphenyl phosphate or diisopropyl phosphorofluoridate (DFP) to hens does not cause clinically observable delayed neurotic effects prior to a point when inhibition of neurotoxic esterase (NTE) of brain and spinal cord reaches 70-90%. These results are contrary to another study with diisopropyl phosphorofluoridate, which claims neuropathy without high inhibition, but are supported by results of chronic feeding experiments with tri-o-cresyl phosphate. The discrepancy cannot be attributed to differences in the strains of hens used.
Title: Biochemical events in delayed neurotoxicity: is aging of chymotrypsin inhibited by saligenin cyclic phosphates a model for aging of neurotoxic esterase? Johnson MK, Clothier B Ref: Toxicol Lett, 5:95, 1980 : PubMed
Chymotrypsin and neurotoxic esterase (NTE) have some similarities. After inhibition of concentrated (80-800 micro M) chymotrypsin by aryl saligenin cyclic phosphates it is known that aging occurs and some phenolic material becomes attached to protein. This binding has now been shown to be a manifestation of non-specific reaction with any available electrophile such as Tris, reduced glutathione (GSH), or protein. The reaction is therefore not a model for the 100% efficient transfer of alkyl groups to protein which occurs during aging of NTE inhibited by dialkyl phosphates.
Title: Repeated small doses of a neurotoxic organophosphate. Monitoring of neurotoxic esterase in brain and spinal cord Lotti M, Johnson MK Ref: Archives of Toxicology, 45:263, 1980 : PubMed
The effects of small repeated oral doses of mono-2-cresyl diphenyl phosphate (MOCP, 2.5 mg/kg/day) on hen brain and spinal cord neurotoxic esterase (NTE) were measured. The enzyme levels were depressed to about 40% and 55% of normal respectively and maintained at that level for 8 weeks. No clinical and only doubtful histological signs of neuropathy were detected. Neuropathy could be precipitated by depressing the level to < 20% either with a single high dose (50 mg/kg), or by an increase of the repeated dose level to 5 mg/kg/day. There was no correlation between inhibition of NTE in the nervous tissue and the "NTE-like" activity in lymphocytes. "NTE-like" activity in spleen was consistently inhibited but to a lesser extent than that in the brain or spinal cord. Brain AChE and BuChE were not affected.
Title: Neurotoxic esterase in human nervous tissue Lotti M, Johnson MK Ref: Journal of Neurochemistry, 34:747, 1980 : PubMed
Title: Clinical and toxicological investigations of a case of delayed neuropathy in man after acute poisoning by an organophosphorus pesticide Hierons R, Johnson MK Ref: Archives of Toxicology, 40:279, 1978 : PubMed
Progressive neuropathy developed in a man during 2--8 weeks after acute poisoning by a pesticide said to contain trichlorphon. The neuropathy was typical of that caused by organophosphorus esters in the delay and in the maintenance of normal conduction velocity in surviving nerve fibres. A sample alleged to be typical of the ingested material was not more active against hen brain neurotoxic esterase (NTE) than was pure trichlorphon. Delayed neuropathy has never been produced in hens by a single dose of trichlorphon. This incident and studies of human brain in vitro suggest that the ratio neurotoxicity/lethality for trichlorphon is higher in man than in the hen. Suggestion is made of laboratory tests to improve neurotoxicity screening.
Title: The anomalous behaviour of dimethyl phosphates in the biochemical test for delayed neurotoxicity Johnson MK Ref: Archives of Toxicology, 41:107, 1978 : PubMed
Several dimethyl phosphate behave anomalously in tests for delayed neurotoxicity. Doses given to hens caused high inhibition of brain neurotoxic esterase (NTE) but no ataxia. Less inhibition of NTE was seen in spinal cord than in brain. Di-isopropyl phosphorofluoridate caused equal inhibition of NTE in brain and cord. When dosing with dimethyl phosphates was repeated NTE inhibition in cord increased and pair-dosed birds became ataxic. In vitro brain and cord NTE were indistinguishable but the in vivo discrepancy between inhibition of brain and cord NTE was matched by a similar discrepancy in inhibition of AChE. It appears that ataxia arises from inhibition of spinal cord NTE and that only in the present cases (among about 200) was the effect in brain not a perfect biochemical monitor.
Title: Neurotoxicity of organophosphorus pesticides: predictions can be based on in vitro studies with hen and human enzymes Lotti M, Johnson MK Ref: Archives of Toxicology, 41:215, 1978 : PubMed
The comparative inhibitory power of organophosphorus esters in vitro against hen brain acetylcholinesterase and neurotoxic esterase correlates with their comparative effects (death or delayed neuropathy) in vivo. Further comparisons of the in vitro effects seen with hen and human enzymes facilitates extrapolations to the human in vivo situation.
Title: Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential Johnson MK Ref: Archives of Toxicology, 37:113, 1977 : PubMed
The assay of neurotoxic esterase (NTE) in brains taken from dosed hens enables potential neurotoxicity of organophosphate pesticides, plasticers, etc. to be assessed. The original assay [Johnson, M.K. Biochem. J. 114, 711-717 (1969)] has been simplified to eliminate centrifugation and transfer steps and both the selectivity and the sensitivity have been increased. The procedures necessary to obtain stable reagent stocks are described.
Title: Side effects of organophosphorus compounds: delayed neurotoxicity Aldridge WN, Johnson MK Ref: Bulletin of the World Health Organization, 44:259, 1971 : PubMed
Title: Studies on delayed neurotoxicity produced by some organophosphorus compounds Aldridge WN, Barnes JM, Johnson MK Ref: Annals of the New York Academy of Sciences, 160:314, 1969 : PubMed
