Rudy J. Richardson

The risk of Parkinson's disease with exposure to pesticides, farming, well water, and rural living

We assessed exposure to pesticides, farming, well water use, and rural living as risk factors for Parkinsons disease (PD) in a population-based case-control study consisting of men and women ≥50 years of age who had primary medical care at Henry Ford Health System in metropolitan Detroit. Enrolled PD patients (n = 144) and control subjects (n = 464) were frequency-matched for age, race, and sex. When adjusted for these variables and smoking status, there was a significant association of occupational exposure to herbicides (odds ratio [OR], 4.10; 95% CI, 1.37, 12.24) and insecticides (OR, 3.55; 95% CI, 1.75, 7.18) with PD, but no relation was found with fungicide exposure. Farming as an occupation was significantly associated with PD (OR, 2.79; 95% CI, 1.03, 7.55), but there was no increased risk of the disease with rural or farm residence or well water use. The association of occupational exposure to herbicides or insecticides with PD remained after adjustment for farming. The association of farming with PD was maintained after adjustment for occupational herbicide exposure and was of borderline significance after adjustment for occupational insecticide exposure. These results suggest that PD is associated with occupational exposure to herbicides and insecticides and to farming and that the risk of farming cannot be accounted for by pesticide exposure alone.

Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: A critical review of the literature

Chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) is a broad-spectrum organophosphorus (OP) insecticide. Anticipated increases in the already extensive use of this compound have prompted this reassessment of its neurotoxicity. Because chlorpyrifos and other OP insecticides are designed to produce acute cholinergic effects through inhibition of acetylcholinesterase (AChE) and some OP compounds can cause OP compound-induced delayed neurotoxicity (OPIDN) via chemical modification of neurotoxic esterase (neuropathy target esterase, NTE), this review focuses on the capacity of chlorpyrifos to precipitate these and other adverse neurological consequences. Chlorpyrifos exhibits only moderate acute toxicity in many mammalian species, due largely to detoxification of the active metabolite, chlorpyrifos oxon, by A-esterases. Rats given large doses of chlorpyrifos (sc in oil) have prolonged inhibition of brain AChE, possibly due to slow release of the parent compound from a depot. Associated cognitive and motor deficits return to normal well before recovery of AChE activity and muscarinic receptor down-regulation, as expected from classic tolerance. Controlled studies of OP compound exposures in humans also indicate that cognitive dysfunction requires substantial AChE inhibition. Information is relatively sparse on neurological dysfunction that is secondary to theoretical reproductive, developmental, or immunological effects, but the best available data indicate that such effects are unlikely to result from exposures to chlorpyrifos. In accord with the much greater inhibitory potency of chlorpyrifos oxon for AChE than for NTE, clinical reports and experimental studies indicate that OPIDN from acute exposures to chlorpyrifos requires doses well in excess of the LD50, even when followed by repeated doses of the OPIDN potentiator phenylmethanesulfonyl fluoride (PMSF). Likewise, studies in hens show that subchronic exposures at the maximum tolerated daily dose do not result in OPIDN. Although exposure to chlorpyrifos as a result of normal use is unlikely to produce classical OPIDN, a recent report stated that mild reversible sensory neuropathy had occurred in eight patients who had been exposed subchronically to unknown amounts of chlorpyrifos. It is not clear whether these cases represent an incorrect linkage of cause and effect, a newly disclosed reversible sensory component of OPIDN, or an entirely new phenomenon. The question of the potential for chlorpyrifos to cause this mild sensory neuropathy could be resolved by the use of quantitative tests of sensory function in animal experiments and/or prospective studies of humans with known exposures to chlorpyrifos.

Neuropathy Target Esterase Gene Mutations Cause Motor Neuron Disease

The possibility that organophosphorus (OP) compounds contribute to motor neuron disease (MND) is supported by association of paraoxonase 1 polymorphisms with amyotrophic lateral sclerosis (ALS) and the occurrence of MND in OP compound-induced delayed neuropathy (OPIDN), in which neuropathy target esterase (NTE) is inhibited by organophosphorylation. We evaluated a consanguineous kindred and a genetically unrelated nonconsanguineous kindred in which affected subjects exhibited progressive spastic paraplegia and distal muscle wasting. Affected subjects resembled those with OPIDN and those with Troyer Syndrome due to SPG20/spartin gene mutation (excluded by genetic linkage and SPG20/spartin sequence analysis). Genome-wide analysis suggested linkage to a 22 cM homozygous locus (D19S565 to D19S884, maximum multipoint LOD score 3.28) on chromosome 19p13 to which NTE had been mapped (GenBank AJ004832). NTE was a candidate because of its role in OPIDN and the similarity of our patients to those with OPIDN. Affected subjects in the consanguineous kindred were homozygous for disease-specific NTE mutation c.3034A-->G that disrupted an interspecies conserved residue (M1012V) in NTEs catalytic domain. Affected subjects in the nonconsanguineous family were compound heterozygotes: one allele had c.2669G-->A mutation, which disrupts an interspecies conserved residue in NTEs catalytic domain (R890H), and the other allele had an insertion (c.2946_2947insCAGC) causing frameshift and protein truncation (p.S982fs1019). Disease-specific, nonconserved NTE mutations in unrelated MND patients indicates NTEs importance in maintaining axonal integrity, raises the possibility that NTE pathway disturbances contribute to other MNDs including ALS, and supports the role of NTE abnormalities in axonopathy produced by neuropathic OP compounds.

Evidence for the existence of neurotoxic esterase in neural and lymphatic tissue of the adult hen

Hen brain and spinal cord contain a number of esterases that hydrolyze phenyl valerate (PV). Most of this activity is sensitive to inhibition by micromolar concentrations of paraoxon. Included among the paraoxon-resistant esterases is neurotoxic esterase (NTE), which is inhibited in vivo and in vitro by certain organophosphorus compounds, such as mipafox, which cause delayed neurotoxicity. Since published information on the NTE content of non-neural tissues was heretofore lacking, a comprehensive study was undertaken of the occurrence of this enzyme in tissues of the adult hen (Gallus gallus domesticus), the species of choice in the study of organophosphorus-induced delayed neurotoxicity. Complete differential titration curves of PV esterase activity were obtained by preincubation of each tissue homogenate with a wide range of concentrations of paraoxon, a non-neurotoxic compound, plus or minus mipafox, a neurotoxic compound, followed by PV esterase assay. Brain NTE activity was determined to be 2426 +/- 104 nmoles.min-1.(g wet weight)-1 (mean +/- S.E.M.). Titration of other tissues resulted in the following NTE activities, expressed as percentages of brain NTE activity: spinal cord (21%), peripheral nerve (1.7%), gastrocnemius muscle (0%), pectoralis muscle (0%), heart (4%), liver (0%), kidney (0%), spleen (70%), spleen lymphocytes (26%), and blood lymphocytes (24%). Using an abbreviated procedure, erythrocytes and plasma showed no NTE activity. These results indicate that NTE has limited distribution among the tissues of the adult hen and is present in lymphatic as well as neural tissue.

SUBCELLULAR DISTRIBUTION OF MARKER ENZYMES AND OF NEUROTOXIC ESTERASE IN ADULT HEN BRAIN

Neurotoxic esterase (NTE) is now regarded as the site of the primary biochemical lesion in the delayed neuronal degeneration produced by certain organophosphorus esters. Since hens are the species of choice in studies of this neuropathy the subcellular distribution of NTE and marker enzymes in adult hen brain was carried out. Up to 70%, of NTE was recovered in a microsomal fraction (P3) which was also enriched in 5′‐nucleotidase (5′‐ribonucleotide phosphohydrolase EC 3.1.3.5), a plasma membrane marker. The protein content of this fraction (31% of the parent homogenate) is double that of equivalent mammalian brain fractions. The LDH distribution suggests that the P3 fraction contained many small synaptosomes. Subfractionation of microsomes by rate and equilibrium centrifugation on sucrose density gradients segregated the RNA but failed to separate the NTE. 5′‐nucleotidase and glucose‐6‐phosphatase (D‐glucose‐6‐phosphate phosphohydrolase EC 3.1.3.9) from each other. NTE was considerably concentrated (2–5 times) in subfractions of the P2 fraction, which are believed to be enriched in synaptosomal membranes. A similar localization of NTE and AChE was found in subfractions of P2 from neonatal chick brain. Axon fragments contained a significant amount of NTE which was not associated with the myelin. Nuclear and mitochondrial fractions were low in NTE. Microsomes could be partitioned in biphasic aqueous polymer systems, but with little enrichment of NTE. The possible association of NTE with synaptosomal membranes suggests that early events in organophosphorus neuropathy may occur at the axonal (? synaptic) surface.

Neuropathy target esterase (NTE): overview and future

Neuropathy target esterase (NTE) was discovered by M.K. Johnson in his quest for the entity responsible for the striking and mysterious paralysis brought about by certain organophosphorus (OP) esters. His pioneering work on OP neuropathy led to the view that the biochemical lesion consisted of NTE that had undergone OP inhibition and aging. Indeed, nonaging NTE inhibitors failed to produce disease but protected against neuropathy from subsequently administered aging inhibitors. Thus, inhibition of NTE activity was not the culprit; rather, formation of an abnormal protein was the agent of the disorder. More recently, however, Paul Glynn and colleagues showed that whereas conventional knockout of the NTE gene was embryonic lethal, conditional knockout of central nervous system NTE produced neurodegeneration, suggesting to these authors that the absence of NTE rather than its presence in some altered form caused disease. We now know that NTE is the 6th member of a 9-protein family called patatin-like phospholipase domain-containing proteins, PNPLA1-9. Mutations in the catalytic domain of NTE (PNPLA6) are associated with a slowly developing disease akin to OP neuropathy and hereditary spastic paraplegia called NTE-related motor neuron disorder (NTE-MND). Furthermore, the NTE protein from affected individuals has altered enzymological characteristics. Moreover, closely related PNPLA7 is regulated by insulin and glucose. These seemingly disparate findings are not necessarily mutually exclusive, but we need to reconcile recent genetic findings with the historical body of toxicological data indicating that inhibition and aging of NTE are both necessary in order to produce neuropathy from exposure to certain OP compounds. Solving this mystery will be satisfying in itself, but it is also an enterprise likely to pay dividends by enhancing our understanding of the physiological and pathogenic roles of the PNPLA family of proteins in neurological health and disease, including a potential role for NTE in diabetic neuropathy.

Lymphocyte and Brain Neurotoxic Esterase: Dose and Time Dependence of Inhibition in the Hen Examined with Three Organophosphorus Esters

Certain organic phosphorus esters produce sensorimotor axonopathy in man and other species. There is an excellent correlation between the capacity of an organophosphorus compound to produce axonopathy and its ability to inhibit brain neurotoxic esterase (NTE) in hens. Because NTE is present in peripheral lymphocytes of both hen and man, it has been suggested that the lymphocyte enzyme might be useful both in experimental and clinical situations as an indicator of exposure to organophosphorus compounds producing axonopathy. Diethyl 4-nitrophenyl phosphate (paraoxon), tri-2-cresyl phosphate (TOCP), methyl 2,5-dichloro-4-bromophenyl phenylphosphonothionate (leptophos), and di-n-butyl-2,2-dichlorovinyl phosphate (di-n-butyl dichlorvos, DBDCV) were used to examine the relationship between lymphocyte and brain NTE inhibition in hens. As expected, paraoxon (0.75 mg/kg) did not inhibit NTE in brain or lymphocytes. TOCP (10 to 100 mg/kg), leptophos (25 to 150 mg/kg), and DBDCV (1.0 to 4.0 mg/kg) inhibited both brain and lymphocyte NTE activity in a dose-related manner with good correlation of inhibition between tissues taken 24 hr after exposure (r2 = 0.53 to 0.67; p less than 0.020 to 0.001). However, correlation of inhibition between tissues taken from animals killed 48 hr after exposures was poor (r2 = 0.15 to 0.30; p less than 0.10 to 0.05), with consistently less inhibition of lymphocyte NTE relative to brain NTE. This study indicates that assay of lymphocyte NTE can provide a good monitor of exposure to axonotoxic organophosphorus compounds within 24 hr between exposure and measurement.

Biomarkers and mechanisms of drug-induced vascular injury in non-rodents.

In preclinical safety studies, drug-induced vascular injury can negatively impact candidate-drug selection because there are no obvious diagnostic markers for monitoring this pathology preclinically or clinically. Furthermore, our current understanding of the pathogenesis of this lesion is limited. While vasodilatation and increased shear stress appear to play a role, the exact mechanism(s) of injury to the primary target cells, smooth muscle (SMC) and endothelial cell (EC), are unknown. Evaluation of potential novel markers for clinical monitoring with a mechanistic underpinning would add value in risk assessment and risk management. This mini review focuses on the efforts and progress to identify diagnostic markers as well as understanding the mechanism of action in nonrodent drug-induced vascular injury.

Neurotoxic esterase: Characterization of the solubilized enzyme and the conditions for its solubilization from chicken brain microsomal membranes with ionic, zwitterionic, or nonionic detergents

Neurotoxic esterase (NTE) is a membrane-bound protein found in highest concentration in brain and lymphocytes. The enzyme has no known physiological function, but its organophosphorylation and aging in neural tissue are thought to trigger the pathogenesis of organophosphorus-induced delayed neuropathy (OPIDN). Solubilization of NTE from microsomal membranes from hen or chick brain was studied with ten detergents encompassing ionic, zwitterionic, or nonionic types. Corrected yields of NTE solubilized over a range of [detergent]/[protein] ratios were determined by dividing the activity not sedimenting in detergent at 100,000 g for 60 min at 4 degrees by the activity in the original microsomal fraction with no detergent present. Highest corrected yields were obtained with sodium cholate (44%), Triton X-100 (48%), and nonyl-GPS (57%). Partial loss of NTE activity occurred in the presence of detergent which could be prevented by the inclusion of asolectin in the solubilization preparation. NTE could not be solubilized by omitting detergent or by substituting 2 M NaCl for detergent. Mipafox pI50 values obtained from complete titration curves carried out on NTE solubilized in Triton X-100, sodium cholate, or sodium cholate/asolectin were indistinguishable from the value for native enzyme from brain homogenate. These results indicate that NTE exhibits the properties of an integral membrane protein with lipid dependence. The enzyme can be solubilized in good yield with a variety of detergents with retention of its characteristic differential inhibition by paraoxon and mipafox, a necessary prelude to bulk purification of the enzymatically active protein.

Thrombin preconditioning provides protection in a 6-hydroxydopamine Parkinson's disease model

Low-dose thrombin given several days before lesioning is neuroprotective in ischemic and hemorrhagic models of stroke, an effect termed thrombin preconditioning (TPC). Here, the ability of TPC to provide protection in a 6-hydroxydopamine (6-OHDA) model of Parkinsons disease (PD) was evaluated. All animals received 10 microg 6-OHDA into the right medial forebrain bundle. Three days prior to 6-OHDA, the animals received either 1 U rat thrombin (n=17) or saline (n=14) 1 mm above the site of neurotoxin delivery. The animals were then evaluated for neurobehavioral deficits until 21 days post-injection. TPC animals performed significantly better on both a vibrissae-elicited forelimb placing test and a forelimb-use asymmetry test than the saline controls. The animals were then sacrificed for either catecholamine determination by HPLC with electrochemical detection or for histopathology to determine lateral ventricular volume or striatal tyrosine hydroxylase immunoreactivity. Although TPC did not protect against the dopamine depletion associated with this severe model, it did reduce dopaminergic terminal loss and ventricular enlargement as compared to saline-treated animals. This report presents the new finding that preconditioning (and TPC in particular) provides protection in a 6-OHDA PD model. Understanding the mechanisms involved in TPC-mediated protection may stimulate innovative therapeutic regimens.