David E. Ray

The potential for toxic effects of chronic, low-dose exposure to organophosphates

Organophosphorus esters have the potential to produce several forms of toxicity. Most produce acute intoxication as a result of inhibition of acetylcholinesterase and, if severe, this can have longer lasting secondary consequences such as intermediate syndrome, or even permanent disability. Some esters produce a very specific syndrome of delayed peripheral neuropathy. This neuropathy is always preceded by severe acute intoxication, except in the case of a few specific agents such as tri-o-cresyl phosphate. All of these effects are reasonably well understood and show a dose threshold. Chronic low level exposure in non-poisoned subjects has been associated with impaired neurobehavioral performance in some, but not all, epidemiological studies. The mechanisms involved are not well understood, but if organophosphates do play a causal role, this will not necessarily be via acetylcholinesterase inhibition. Doses too low to produce cholinergic signs have been shown to produce a variety of effects in experimental animals ranging from enhanced maze learning to slowed nerve conduction. It is likely that other, more sensitive, brain proteins are the targets for such actions. Effects mediated via such target proteins would be expected to show very different structure-activity relationships to acute toxicity mediated by acetylcholinesterase. Hence epidemiological studies expecting similar (class) effects from low-dose exposure to different organophosphorus esters may produce variable results or false negatives.

Pyrethroid Insecticides: Poisoning Syndromes, Synergies, and Therapy

Background: Pyrethroid insecticides are widely used, but there have been relatively few reports of systemic poisoning. These reports have, however, shown that pharmacotherapy is difficult and that the duration of poisoning can be unexpectedly long. Pyrethroids are ion channel toxins prolonging neuronal excitation, but are not directly cytotoxic. Two basic poisoning syndromes are seen. Type I pyrethroids produce reflex hyperexcitability and fine tremor. Type II pyrethroids produce salivation, hyperexcitability, choreoathetosis, and seizures. Both produce potent sympathetic activation. Local effects are also seen: skin contamination producing paresthesia and ingestion producing gastrointestinal irritation. The slow absorption of pyrethroids across the skin usually prevents systemic poisoning, although a significant reservoir of pyrethroid may remain bound to the epidermis. Carboxyesterase inhibitors can enhance pyrethroid toxicity in high-dose experimental studies. Hence, the unauthorized pyrethroid/organophosphate mixtures marketed in some developing countries may precipitate human poisoning. Pyrethroid paresthesia can be treated by decontamination of the skin, but systemic poisoning is difficult to control with anticonvulsants. Pentobarbitone, however, is surprisingly effective as therapy against systemic type II pyrethroid poisoning in rats, probably due to its dual action as a chloride channel agonist and a membrane stabilizer.

Evidence for a separate mechanism of toxicity for the Type I and the Type II pyrethroid insecticides

Neurotoxicity and mechanistic data were collected for six alpha-cyano pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin and lambda-cyhalothrin) and up to six non-cyano containing pyrethroids (bifenthrin, S-bioallethrin [or allethrin], permethrin, pyrethrins, resmethrin [or its cis-isomer, cismethrin] and tefluthrin under standard conditions. Factor analysis and multivariate dissimilarity analysis were employed to evaluate four independent data sets comprised of (1) fifty-six behavioral and physiological parameters from an acute neurotoxicity functional observatory battery (FOB), (2) eight electrophysiological parameters from voltage clamp experiments conducted on the Na(v)1.8 sodium channel expressed in Xenopus oocytes, (3) indices of efficacy, potency and binding calculated for calcium ion influx across neuronal membranes, membrane depolarization and glutamate released from rat brain synaptosomes and (4) changes in chloride channel open state probability using a patch voltage clamp technique for membranes isolated from mouse neuroblastoma cells. The pyrethroids segregated into Type I (T--syndrome-tremors) and Type II (CS syndrome--choreoathetosis with salivation) groups based on FOB data. Of the alpha-cyano pyrethroids, deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin arrayed themselves strongly in a dose-dependent manner along two factors that characterize the CS syndrome. Esfenvalerate and fenpropathrin displayed weaker response profiles compared to the non-cyano pyrethroids. Visual clustering on multidimensional scaling (MDS) maps based upon sodium ion channel and calcium influx and glutamate release dissimilarities gave similar groupings. The non-cyano containing pyrethroids were arrayed in a dose-dependent manner along two different factors that characterize the T-syndrome. Bifenthrin was an outlier when MDS maps of the non-cyano pyrethroids were based on sodium ion channel characteristics and permethrin was an outlier when the MDS maps were based on calcium influx/glutamate release potency. Four of six alpha-cyano pyrethroids (lambda-cyfluthrin, cypermethrin, deltamethrin and fenpropathrin) reduced open chloride channel probability. The R-isomers of lambda-l-cyhalothrin reduced open channel probability whereas the S-isomers, antagonized the action of the R-isomers. None of the non-cyano pyrethroids reduced open channel probability, except bioallethrin, which gave a weak response. Overall, based upon neurotoxicity data and the effect of pyrethroids on sodium, calcium and chloride ion channels, it is proposed that bioallethrin, cismethrin, tefluthrin, bifenthrin and permethrin belong to one common mechanism group and deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin belong to a second. Fenpropathrin and esfenvalerate occupy an intermediate position between these two groups.

Focal astrocyte loss is followed by microvascular damage, with subsequent repair of the blood-brain barrier in the apparent absence of direct astrocytic contact.

Blood‐brain barrier (BBB) breakdown is a feature of cerebral ischaemia, multiple sclerosis, and other neurodegenerative diseases, yet the relationship between astrocytes and the BBB integrity remains unclear. We present a simple in vivo model in which primary astrocyte loss is followed by microvascular damage, using the metabolic toxin 3‐chloropropanediol (S‐α‐chlorohydrin). This model is uncomplicated by trauma, ischaemia, or primary immune involvement, permitting the study of the role of astrocytes in vascular endothelium integrity, maintenance of the BBB, and neuronal function. Male Fisher F344 rats given 3‐chloropropanediol show astrocytic damage and death at 4–24 h in symmetrical brainstem and midbrain nuclear lesions, while neurons show morphological changes at 24–48 h. Fluorescent 10 kDa dextran tracers show the BBB leaking from 24 h, progressing to petechial haemorrhage after 48–72 h, with apparent repair after 6 days. BBB breakdown, but not the earlier astrocytic death, is accompanied by a delayed increase in blood flow in the inferior colliculus. An ED1 inflammatory response develops well after astrocyte loss, suggesting that inflammation may not be a factor in starting BBB breakdown. This model demonstrates that the BBB can self‐repair despite the apparent absence of direct astrocytic‐endothelial contact. The temporal separation of pathological events allows pharmacological intervention, and the mild reversible ataxia permits long‐term survival studies of repair mechanisms.

Chronic effects of low level exposure to anticholinesterases — a mechanistic review

High dose exposure to anticholinesterases which results in symptomatic poisoning can have lasting consequences due to the trauma of intoxication, excitotoxicity, secondary hypoxic damage, and (for some agents) a delayed onset polyneuropathy (OPIDN). The potential effects of low level exposure are less well defined. The most reliable data comes from controlled clinical trials with specific agents. A single dose of sarin or repeated doses of metrifonate or mevinphos, have produced only transient adverse effects at doses causing substantial acetylcholinesterase inhibition. Other data comes from epidemiological surveys. These have often used more sensitive indices than the clinical studies, but are less reliable due to the difficulty of defining exposure and matching control and exposed populations. Subtle, mainly cognitive, differences between exposed and non-exposed populations are sometimes seen. Low level exposure can cause a reversible down-regulation of cholinergic systems, and a range of non-cholinesterase effects that are structure-specific, and do not always parallel acute toxicity. Novel protein targets sensitive to low level exposure to some organophosphates are known to exist in the brain, but their functional significance is not yet understood.

Reversible disruption of tight junction complexes in the rat blood-brain barrier, following transitory focal astrocyte loss

Breakdown of the blood‐brain barrier is a feature of acute and chronic neurodegenerative changes, yet the relationship between astrocytes and the mature barrier remains unclear. We studied this role of astrocytes in vivo using a gliotoxin and evaluated changes in three vascular tight junction markers. Male Fisher F344 rats given systemic 3‐chloropropanediol showed astrocytic loss in the inferior colliculus from 12–24 h until the lesion was repopulated 8–28 days later. Within 6 h of astrocyte loss, microvessels in this area began to demonstrate a loss of the normal paracellular localization of the transmembrane proteins occludin and claudin‐5 and cytoplasmic zonula occludens‐1, which correlated with focal vascular leak of dextran (10 kDa) and fibrinogen. Platelet endothelial adhesion molecule‐1 staining revealed that there was no loss of the endothelial lining. Between 4–8 days, severe downregulation of tight junction protein expression was observed, which subsequently returned over the same time period as astrocytes repopulated the lesion. Unexpectedly, dextran and fibrinogen leak from vessels had ceased at 6 days, well before the return of occludin and claudin‐5 to appropriate paracellular domains. Control nonvulnerable cortical tissue showed no change in astrocyte morphology and tight junction expression over the same time course. Our data supports a primary role for astrocytic contact in the expression of occludin, claudin‐5, and zonula occludens‐1 in the mature brain vasculature in vivo. However, barrier integrity to dextran (10 kDa) and fibrinogen can be restored in the absence of astrocytes and tight junction proteins (occludin, claudin‐5, and zonula occludens‐1).

A study of the kinetic behaviour of glucose based on simultaneous estimates of influx and phosphorylation in brain regions of rats in different physiological states

Results from the application of a procedure that allows simultaneous estimation of the rates of glucose transport from blood and of glucose phosphorylation in discrete regions of the brain are given for groups of rats displaying chemically induced motor disturbances. The procedure is based on sequential injections of [14C]- and [3H]-2-deoxyglucose followed a few minutes later by focused microwave irradiation to the head. Control conscious rats were used and rats displaying either whole body tremors, hind-limb rigidity or choreoathetotic movements induced by synthetic pyrethroid compounds. For individual rats estimates of 5 parameters were obtained in up to 16 brain regions. In addition brain tissue and plasma glucose concentrations were determined. The 5 parameters were: (1) rate of total glucose influx from plasma; (2) rate of glucose phosphorylation (equivalent to net influx); (3) rate of glucose efflux; (4) half-life of free glucose in brain; and (5) PS-product expressed as the ratio of the rate of glucose influx to plasma glucose concentration. For each parameter significant differences between regions were found in all groups of animals including conscious controls. The mean values for the somatosensory cortex of control rats were parameter: (1) 2.02 mumol/g/min; (2) 1.11 mumol/g/min; (3) 0.91 mumol/g/min; (4) 0.94 min; and (5) 0.196 ml/g/min. A high correlation was observed between the rate of total glucose influx and the rate of glucose phosphorylation for all brain regions in all groups of rats. This finding is discussed in terms of a synchronized regulatory mechanism on the glucose transport carrier of capillary endothelial cells and on the functional hexokinase Vmax activity within brain cells.

Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Novel protein targets for organophosphorus compounds

Inhibition of tritiated di-isopropyl phosphorofluoridate labelling by a range of organophopshorus compounds was used to screen for novel OP-reactive targets in rat-brain homogenates. Analysis of target proteins was conducted by SDS/PAGE and detection of tritiated proteins using a thin layer chromatography (TLC) linear analyser. Two major sites of 3H-DFP labelling were found with relative molecular masses of 30 and 85 kDa. Rates of reaction of these labelling sites with a range of OP compounds were compared to that of acetylcholinesterase. The 30 kDa band was found to be more sensitive to paraoxon, dichlorvos and diazoxon than acetylcholinesterase. The 85 kDa band was found to be more sensitive to dichlorvos and diazoxon than acetylcholinesterase. Neither labelling band reacted with chlorfenvinphos or demeton-s-methyl at significant rates.

Vascular factors in the neurotoxic damage caused by 1, 3-dinitrobenzene in the rat

Using a 3 times 10 mg/kg dose schedule of 1, 3‐dinitrobenzene (DNB) over two days in Fischer rats, we have found the following changes in vascular function and structure during the early phase of the symmetrical brain stem lesions.