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有机磷农药毒理学的研究现状

Current issues in organophosphate toxicology

Lucio G. Costa
Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt Way NE, Suite 100 Seattle, WA 98105, USA
Department of Human Anatomy, Pharmacology and Forensic Sciences, University of Parma, Italy

Abstract

Organophosphates (OPs) are one of the main classes of insecticides, in use since the mid 1940s. OPs can exert significant adverse effects in non-target species including humans. Because of the phosphorylation of acetylcholinesterase, they exert primarily a cholinergic toxicity, however, some can also cause a delayed polyneuropathy. Currently debated and investigated issues in the toxicology of OPs are presented in this review. These include: 1) possible long-term effects of chronic low-level exposures; 2) genetic susceptibility to OP toxicity; 3) developmental toxicity and neurotoxicity; 4) common mechanism of action; 5) mechanisms of delayed neurotoxicity; and 6) possible additional OP targets. Continuing and recent debates, and molecular advances in these areas, and their contributions to our understanding of the toxicology of OPs are discussed.

Keywords: Organophosphates; Acetylcholinesterase; Neuropathy target esterase; Low chronic exposure; Genetic susceptibility; Developmental neurotoxicity; Common mechanism of action; Non-acetylcholinesterase targets

1. Organophosphorus: insecticides

Although a number of organic phosphorus (OP) compounds were synthesized in the 1800s, their development as insecticides only occurred in the late 1930s and early 1940s. The German chemist Gerhard Schrader is credited for the discovery of the general chemical structure of anticholinesterase OP compounds and for the synthesis of the first commercialized OP insecticide [Bladan, containing TEPP (tetraethyl pyrophosphate) as an active ingredient], and for one of the most known, parathion, in 1944 [1]. Since then, hundreds of OP compounds have been made and commercialized worldwide in a variety of formulations.

The chemistry of OPs, which leads to their classification in several subclasses, has been thoroughly investigated [2]. The general structure of OP insecticides can be represented by

Where X is the so-called “leaving group”, that is displaced when the OP phosphorylates acetylcholinesterase (AChE), and is most sensitive to hydrolysis; R1 and R2 are most commonly alkoxy groups, through other chemical substitutes are also possible; either an oxygen or sulfur atom are also attached to the phosphorus with a double bond.

The complex array of reactions involved in the biotransformation of OPs in target and non-target species has also been the subject of extensive investigations [3]. For compounds that contain a sulfur bound to the phosphorus, a metabolic bioactivation is necessary for their main biological activity to be manifest, as only compounds with a P = O moiety are effective inhibitors of AChE. This bioactivation thus consists in an oxidative desulfuration mediated, mostly but not exclusively in the liver, by cytochromes P450 enzymes (CYPs), and leading to the formation of an “oxon” or oxygen analog of the parent insecticide (Fig. 1). Though this reaction has been known for several decades, the exact CYP isoforms(s) involved are still elusive, but available data suggests that the overall picture is quite complex. Multiple CYPs have shown to bioactivate organophosphorothioates to their oxons, with different substrate specificities. For example, diazinon is activated by human hepatic CYP2C19 [4], while parathion is activated primarily by CYP3A4/5 and CYP2C8 [5]. The CYPs can also catalyze several oxidation, and a few reduction, reactions that lead to detoxication of OP compounds. Some of these are shown in Fig. 1 for the OP insecticide parathion. Differences between CYPs exist also in the detoxication process. For example, while CYP2B6 metabolizes chlorpyrifos primarily to the oxon, it metabolizes parathion primarily to p-nitrophenol [6].

Fig. 2. Schematic representation of biochemical interactions between OPs and AChE. Reaction 1 leads to phosphorylated AChE. Reaction 2 is spontaneous reactivation of AChE. The rate of this reaction can be accelerated by oximes. Reaction 3 is the aging and leads to a stable, negatively charged phosphorylated AChE. E-OH, active site of the enzyme.

2. AChE and NTE as primary targets

The primary target for OPs is AChE, a B-esterase whose physiological role is that of hydrolyzing acetylcholine, a major neurotransmitter in the peripheral (autonomic and motor–somatic) and central nervous systems. OPs with a P = O moiety phosphorylate an hydroxyl group on serine in the active (esteratic) site of the enzyme, thus impeding its action on the physiological substrate (Fig. 2). The bond between the phosphorus atom and the esteratic site of the enzyme is much more stable than the bond between the carbonyl carbon of acetate (in acetylcholine) at the same enzyme site. While breaking of the carbon–enzyme bond is complete in a few microseconds, breaking the phosphorus–enzyme bond can take from a few hours to several days, depending on the chemical structure of the OP [1]. Phosphorylated AChE is hydrolyzed by water at a very slow rate, depending, as said, from the chemical nature of the R substituents (e.g., reactivation decreases in the order demethoxy > diethoxy   diisopropoxy). While water is a weak nucleophilic agent, certain hydroxylamine derivatives, known as oximes, contain a positively charged atom capable of attaching to the anionic site of AChE, and facilitate desphosphorylation of the enzyme. These reactivators are utilized in the therapy of OP poisoning.

Reactivation of phosphorylated AChE does not occur once the enzyme–inhibitor complex has “aged” (Fig. 2). Aging consists in the loss (by nonenzymatic hydrolysis) of one of the two alkoxy (R) groups, and the rate of aging depends on the nature of the alkoxy group (e.g., AChE phosphorylated by an isopropoxy phosphate ages most rapidly). When phosphorylated AChE has aged, the enzyme can be considered to be irreversibly inhibited, and the only means of replacing its activity is through synthesis of new enzyme, a process that could take days.

Acetylcholine released from cholinergic nerve terminals is disposed of solely through hydrolysis by AChE. In fact, differently from other neurotransmitters (e.g., noradrenaline), it is the product of acetylcholine hydrolysis by AChE, choline, that is taken up by the presynaptic terminal. Hence, inhibition of AChE by OPs causes accumulation of acetylcholine at cholinergic synapses, with over-stimulation of muscarinic and nicotinic receptors. This “cholinergic syndrome” includes increased sweating and salivation, profound bronchial secretion, bronchoconstriction, miosis, increased gastrointestinal motility, diarrhea, tremors, muscular twitching and various central nervous system effects. When death occurs, this is believed to be due to respiratory failure due to inhibition of respiratory centers in the brainstem, bronchoconstriction and increased bronchial secretion, and flaccid paralysis of the respiratory muscles [8] and [9]. In addition to the aforementioned oximes, which reactivate phosphorylated AChE before aging has occurred, pharmacological treatment of OP poisoning involves the use of atropine, an acetylcholine antagonist that prevents the action of acetylcholine on muscarinic receptors. Additional pharmacological (e.g., diazepam) and supportive (e.g., artificial ventilation) treatments are also utilized in case of severe poisoning [9].

The early discovery of AChE inhibition by OP compounds has led to their development primarily as insecticides, but also as nerve agents and to a limited extent, as drugs. OPs are potent and effective insecticides and still represent the largest group of insecticides sold worldwide. Given the strong similarities of the insect and mammalian cholinergic nervous system, these compounds are, however, responsible for the million of poisonings and thousands of deaths occurring annually as a result of pesticide exposures, particularly in third world countries. Certain OPs, in particular those containing isopropoxy substituents, have also been developed, and some have been used, as nerve agents for chemical warfare. Examples of such compounds are soman, sarin, tabun and VX. A few OPs were also developed as pharmaceutical drugs. Examples are metrifonate (trichlorfon), used on anthelmintic in schistosamiasis, or echothiophate, used for the treatment of glaucoma. However, use of AChE inhibitors for treatment of these and other conditions (e.g., myasthenia gravis, termination of the effects of competitive neuromuscular blockers, Alzheimer‘s disease) relies mostly on carbamates or other compounds [10].

A few OPs can also cause another type of toxicity known as organophosphate-induced delayed polyneuropathy (OPIDP). Signs and symptoms include tingling of the hands and feet, followed by sensory loss, progressive muscle weakness and flaccidity of the distal skeletal muscles of the lower and upper extremities, and ataxia [11], [12] and [13]. These occur starting several days (usually 2–3 weeks) after a single exposure, when both cholinergic and intermediate (see below) syndrome signs have subsided. OPIDP can be classified as a distal sensorimotor axonopathy. Neuropathological studies in experimental OPIDP have evidenced that the primary lesion is a bilateral degenerative change in distal levels of axons and their terminals, primarily affecting larger/longer myelinated central and peripheral nerve fibers, leading to breakdown of neuritic segments and of their myelin sheats [12].

OPIDP is not related to AChE inhibition. Indeed, one of the compounds involved in several epidemics of this neuropathy (TOCP, tri-ortho-cresyl phosphate) is a very poor AChE inhibitor. Extensive studies carried out in the past 30 years [11], [14] and [14a] have identified the target in another esterase, named neuropathy target esterase (NTE). Several OPs, depending on their chemical structure, can inhibit NTE, as do some non-OPs, such as carbamates and sulfonyl fluorides. Phosphorylation of NTE by OPs is similar to that observed for AChE. However, only OPs whose chemical structure leads to aging of phosphorylated NTE (by a process analogous to that described for AChE) can cause OPIDP. Other compounds that inhibit NTE but cannot undergo the aging reaction are not neuropathic, indicating that inhibition of NTE catalytic activity is not the mechanism of axonal degeneration. For OPIDP to be initiated, phosphorylation of NTE and subsequent aging of at least 70% of NTE is necessary, and this two-step process occurs within hours of poisoning. Yet, the first clinical signs are only evident weeks later, when NTE activity has recovered. When given to experimental animals before a neuropathic OP, agents that inhibit NTE but do not age, exert a protective role, by occupying the NTE active site. However, when given after a neuropathic OP, these compounds promote OPIDP [15]. As there is compelling evidence that phosphorylation and aging of NTE is essential for the development of OPIDP, compounds that do so have been named initiators. Other inhibitors of NTE that do not initiate OPIDP but that can promote it, have been names promoters, utilizing a terminology used for carcinogenic compounds [16]. Why these compounds would be protective when given before an OP initiator is easy to understand; however, how they are able to promote OPIDP is still unknown. To further complicate this issue, compounds that act as promoters have been shown to potentiate axonal degeneration caused by means other than OPs, such as traumatic nerve lesion or 2,5-hexanedione [15] and [17]. Moreover, promotion has been shown to occur, in some cases, even when the promoter, at doses that do not inhibit NTE, was given before the initiator of OPIDP, suggesting that the mechanism involved in promotion is present and active in healthy axons [17]. Since promotion is less efficient in chicks, where the compensation/repair mechanisms are thought to be more efficient, the current hypothesis is that promotion may directly affect compensation/repair mechanisms(s) of the nervous system [15].

Though it was once thought that some animal species (e.g., rodents) were insensitive to OPIDP, only mice appear to be somewhat resistant [18]. Age, on the other hand, is an important determinant of susceptibility, with young animals displaying more resistance (e.g., in young chicks the threshold for NTE inhibition and aging is > 90% vs. 70% in adult hens; [19]). Hypotheses to explain the consequences of these OP-NTE interactions include a loss of non-esterase functions of NTE essential for the axon, or a gain of toxic function of phosphorylated/aged NTE [13]. Though reductions in axonal transport have been found to precede overt clinical signs, the exact chain of events occurring between phosphorylation and aging of NTE and axonal degeneration remain obscure. Similarly, the physiological function(s) of NTE are not yet known, though recent studies (see Section 3.5) have attempted to clarify it.

Through several epidemics of OPIDP have occurred in the past, its occurrence is now rare. Before commercialization, OPs must undergo specific neurotoxicity testing in the hen (one of the most sensitive species) to determine whether OPIDP is produced. High doses of OPs are used, and animals are protected from acute cholinergic toxicity with atropine, and clinical, morphological and biochemical measurements are carried out. In vitro tests can provide the ratio of relative inhibitory potency toward AChE and NTE, but these have not been accepted by regulatory agencies [20]. Despite these tests, a few commercialized OPs (methamidophos, trichlorfon, chlorpyrifos) have caused OPIDP in humans, mostly as a result of extremely high exposure in suicide attempts [13].

A third clinical manifestation of OP toxicity, the so-called intermediate syndrome, should also be briefly discussed. The intermediate syndrome is characterized by weakness of respiratory, neck and proximal limb muscles [21]. It is not a direct effect of AChE inhibition, and appears several hours after the beginning of signs and symptoms of severe cholinergic over-stimulation, but before eventual signs of OPIDP (hence the name intermediate). The intermediate syndrome is seen in 20–50% of acute OP poisoning cases, where it develops during recovery from cholinergic manifestations. The underlying mechanisms are unknown, but an hypothesis is that muscle weakness may result from cholinergic receptor desensitization due to prolonged cholinergic stimulation [9].

3. Continuing issues in OP toxicology

Despite having been around for over sixty years with an almost as long knowledge of their main mechanism of action and adverse health effects, OPs continue to be the subject of much research efforts. For example, a Medline search (August 2005) with the terms organophosphate/organophosphorus provided around 5000 hits since the year 2000, more than one fourth of all citations since the mid 1950s. The reasons for these research activities, in addition to the continuing high worldwide use of these compounds, lies in the fact that several issues related to their adverse health effects, though debated, at least in some cases, for quite some time, have not been adequately resolved or addressed. Some of the current issues in OP toxicology are listed in Table 1. Below, these issues are briefly discussed, to underscore the fact that a number of questions posed in Table 1, still require satisfactory answers. The discussion is by no means comprehensive or exhaustive but only acknowledges main points of contention or debate, with emphasis on areas where molecular advances have occurred.

Table 1. Some continuing issues in organophosphate toxicology

3.1. Low chronic exposure

While the acute effects of OP exposure have been, for the most part, clearly identified and characterized by thousands of animal studies and cases of human poisonings, the debate is still on two issues: 1) Does acute exposure to an OP, which produces cholinergic signs of toxicity, result in long-term adverse health effects, particularly at the level of the CNS?; and 2) Does chronic low exposure to OPs, at doses that produce no cholinergic signs, produce long-term adverse health effects, particularly in the CNS? With regard to question 1, the current prevalent view is that acute exposure to high doses of OPs may be associated with certain long-term adverse health effects [22], [23] and [24]. Indeed, animal studies have shown that a single high acute exposure to an OP can cause long-lasting behavioral effects [25] and [26], and this has been seen in several human studies as well (e.g., [27] and [28]). Question 2 is still open, as evidence describing long-term neuropsychological or neuropsychiatric alterations upon low chronic exposure is contradictory [29], [30], [31] and [32]. Chronic exposure of animals to OPs usually results in the development of tolerance to their cholinergic effects, which is mediated, at least in part, by down-regulation of cholinergic receptors [33]. However, such exposure can also be associated with neurobehavioral abnormalities, particularly at the cognitive level (e.g., [34] and [35]), though they are usually associated with significant inhibition of AChE. Most recent expert reviews tend to conclude that the balance of evidence does not support the existence of clinically significant neuropsychological effects, neuropsychiatric abnormalities, or peripheral nerve dysfunction in humans chronically exposed to low levels of OPs [23], [36], [37], [38] and [39]. Yet, research in this area will undoubtedly continue, and may benefit from a better understanding of potential noncholinergic effects of OPs (see Section 3.6), which may offer biological plausibility for at least some of the observed effects.

3.2. Genetic susceptibility

Genetically determined variations in biotransformation enzymes, target molecules or cellular repair processes can modify the response to exogenous agents including OPs. This area of research on gene-environment interactions, known as ecogenetics, is aimed at identifying genetic polymorphisms that may modify the risk for adverse health effects upon exposure to a xenobiotic [40], [41] and [42]. In case of OPs, genetic polymorphisms in biotransformation enzymes or target molecules can affect their toxicity [43] and [44]. As discussed earlier, CYPs are important for the activation and detoxication of OP insecticides. Variant forms of several CYP genes have been identified, and these polymorphisms confer differences in catalytic activity or level of expression, which may result in varying rates of oxidation of xenobiotics among individuals [45]. However, as information on the role of specific CYP isozymes in the metabolism of OPs is just beginning to emerge, very limited information is available on the potential contribution of such genetic polymorphisms to OP toxicity susceptibility.

Certain OPs, such as the oxygen analogs of commonly used insecticides (chlorpyrifos oxon, diazoxon, paraoxon) and nerve agents, such as sarin or soman, can be hydrolyzed by paraoxonase (PON1) [7]. The PON1 gene presents several polymorphisms in the coding and promoter regions that affect the catalytic efficiency of the enzyme toward different substrates (the Q192R polymorphism) and its level of expression (e.g., the C-108T polymorphism) [46] and [47]. Extensive research in transgenic animal models clearly indicates that PON1 “status”, encompassing both the Q192R polymorphism and the level of PON activity, plays a most relevant role in modulating the acute toxicity of some, but not all OPs [48] and [49]. The important determinant is the catalytic efficiency of each PON1 allozyme toward a specific substrate; thus, in case of chlorpyrifos oxon, PON1 provides protection in vivo, and PON1_R192 provides better protection than PON1_Q192; in case of diazoxon, both alloforms provide the same degree of protection, while in case of paraoxon, the substrate after which the enzyme was named, PON1 does not provide any protection due to an overall low catalytic efficiency of PON1 toward this substrate [48] and [49] (Table 2). These studies in transgenic mice provide a convincing case of extrapolating the results obtained in animals to humans; however, direct and conclusive confirmation of the relevance of PON1 status in determining relative susceptibility to OP toxicity is still lacking [50] and [51]. This too is expected to be a fruitful area of future research.

Table 2. In vivo protection of human PON1₁₉₂ alloforms and in vitro catalytic efficiency

Data are adapted from Li et al. [48] and Cole et al. [49].
^a V_max/km, measured in vitro under physiological NaCl conditions.
^b Brain AChE activity (fold increase) following challenges with the OPs in PON1 knockout mice pretreated with equal amount of PON1_Q192 or PON1_R192 compared to untreated PON1 ?/? mice.
^c Brain AChE activity (fold increase) following challenge with CPO of transgenic mice expressing the human PON_192R or PON1_192Q alloform over a knockout background, compared to PON1 knockout mice.

Butyrylcholinesterase (BChE), often referred to as pseudocholinesterase or cholinesterase, is a B-esterase synthesized in the liver and secreted in the plasma, whose physiological role is still unclear, though it hydrolyzes a variety of choline esters and many drugs, including drugs of abuse. OPs can bind to BChE and inhibit its activity. High levels of BChE are present in plasma; by scavenging OPs, BChE can guard against their toxicity, as the OP would be unavailable for reaction with the primary target AChE [44]. A large number of genetic polymorphisms have been described for BChE. In addition to the wild-type allele, there are at least 39 identified genetic variants with nucleotide alterations in the coding region [52]. Several of the these variants are silent (i.e., they have 0–10% of normal activity), but they are rare; most common variants (e.g., atypical variants) have a reduced activity and are far less efficient scavengers of positively charged cholinesterase inhibitors [52]. Individuals with genetic variants of BChE with no or low activity would be predicted to be more susceptible to OP toxicity. A study in Brazilian farmers supports this hypothesis [53] but additional investigations are certainly warranted.

To date, only one genetic variant of human AChE has been described, which is not associated with any abnormality in activity [54]. Based on work in AChE knockout mice, it has been suggested, however, that silent AChE alleles may exist in humans [54a]. This work on AChE in transgenic mice models is providing interesting and important insights on the roles of AChE and BChE in OP toxicity. Targeted deletion of four exons in the AChE gene, which totally eliminated AChE activity in nullizygous mice, was not lethal, but AChE^?/? mice displayed a number of physical, behavioral and biochemical abnormalities [55], [56] and [57]. BChE activity was similar in AChE^?/?, wild-type and AChE^+/? mice; differently from AChE^?/? animals, the latter had normal appearance and development, and were fertile [55] and [58]. AChE knockout mice had 50–80% less muscarinic receptors (M₁, M₂ and M₄) in the hippocampus, and decreased muscarinic receptor signaling [59]. These effects are also seen in the development of tolerance to OPs, and may be due to receptor down regulation due to overstimulation by acetylcholine. AChE^?/? mice share some characteristics with a wild-type mouse intoxicated with an OP. These include loss of AChE catalytic activity, body tremors, muscle weakness, reduced pain response, pinpoint pupils and increased salivation and lacrimation [60]. However, a 100% inhibition of AChE by an OP, normally results in lethality. Hence, in AChE^?/? mice, other enzymes must take over in hydrolyzing, albeit partially, acetylcholine. This role has been shown to be fulfilled by BChE, which in AChE knockout mice can hydrolyze acetylcholine in the central and peripheral nervous systems [58] and [61]. The BChE inhibitor bambuterol was indeed lethal to AChE^?/? mice, while it had no effect in wild-type animals [55]. The presence of BChE in mammals may explain why deletion of AChE in lethal in Drosophila, where there is no BChE [62].

Of interest is the response to OPs of AChE^?/? mice that lack their primary target. These animals are supersensitive to the acute toxicity of diisopropylfluorophosphate (DFP) and of VX [55] and [60], as well as of chlorpyrifos oxon [57] (Table 3). The signs of toxicity upon VX administration were identical in wild-type and AChE^?/? mice, indicating that they were due to cholinergic hyperstimulation. This may be explained by the fact that VX inhibited also BChE; however, inhibition was only partial. Furthermore, atropine protected wild-type mice, but not AChE^?/? mice from VX toxicity [60]. Why is then the AChE^?/? mouse supersensitive to OP toxicity? Inhibition of BChE appears to be the primary explanation. However, the possibility that additional targets for OPs may be involved has also been raised [57] and [60] (see Section 3.6). Finally, it should be noted that mice carrying only one deficient AChE allele (AChE^+/?) are healthy, but display an intermediate supersensitivity to OPs [57] and [60] (Table 3). The existence of genetic polymorphisms of AChE in humans that would cause partial AChE deficiency has not been yet demonstrated, but has been hypothesized to exist [57]. These individuals would be healthy, but would display increased susceptibility to OP toxicity.

Table 3.  Sensitivity of AChE deficient mice to OP toxicity

Doses are LD50, sc, and are expressed in mg/kg.

DFP, Diisopropylfluorophosphate; CPO, chlorpyrifos oxon; VX, O-ethyl-S-[2-(Diisopropylamino) ethyl] methylphosphonothioate.     Adapted from Lockridge et al. [57]. 

3.3. Developmental toxicity and neurotoxicity

A continuous priority in public health has been the protection of children from adverse health effects of environmental toxicants, and the removal of lead from gasoline because of its developmental neurotoxicity can be seen as a main event of this endeavor. There is now recognition that children are not just little adults, but there exist child-specific factors that may render them more susceptible to environmental exposures; these include both toxicokinetic and toxicodynamic components, particularly at the level of the CNS [63]. In the past decade, a report from the National Academy of Sciences has highlighted the potential higher exposure of children to pesticides [64], and the Food Quality Protection Act indicates that in the risk assessment process, an additional safety factor should be included to ensure protection of children who are presumed to be more sensitive to toxicants‘ effects [65].

In case of OPs, the issues are whether the young may be more susceptible to the acute toxicity of OPs (AChE inhibition), and whether OPs may cause developmental neurotoxicity. Experimental data indicate that the acute toxicity of OPs is influenced by age, with young animals being more sensitive to the effects of exposure [66] and [67]. Indeed, when comparing lethal dose levels or maximum tolerated doses (MTD) of various OPs, young rats have consistently been shown to be more sensitive (Table 4). This increased sensitivity is not due to intrinsic differences in AChE, whose catalytic properties are not influenced by age [67], but rather to lower metabolic abilities of young animals. For example, in case of parathion and methylparathion, low detoxication by CYPs appeared to be most relevant for the age-dependent susceptibility [68]. In case of chlorpyrifos and diazinon, a lower hydrolytic detoxication by PON1, and perhaps carboxylesterase, accounts for the differential age-related in their acute toxicity [69] and [70]. As these enzymatic systems are believed to show a developmental curve also in humans (see for example Ref. [71] with regard to PON1), young children would be expected to be more sensitive than adults to acute OP toxicity. On the other hand, with regard to OPIDP, the young appear to be more resistant [19] and [72]. Furthermore, these is also some evidence suggesting that enhanced sensitivity to AChE inhibition may not extend to situations of repeated sublethal exposures to OPs [73].

Table 4.  Acute toxicity of OPs in neonatal and adult rats

Data from: ^aBenke and Murphy, 1975 [68]; ^bHarbison, 1975 [120]; ^cBrodeur and Dubois, 1963 [121]; ^dZheng et al. 2000 [122]; ^eMendoza, 1976, [123]; ^fPope et al. 1991 [124].
¹ LD50 and MTD are expressed in mg/kg. Route of administration was oral or ip.
² Neonatal rats are at postnatal days 1–8.

An increasing body of literature suggests that developmental exposure to OPs (though most work has been carried out with a single compound, chlorpyrifos), at dose levels causing little or no inhibition of AChE, results in biochemical and behavioral abnormalities. Experimental studies in rodents indicate that pre- or postnatal exposure to chlorpyrifos affects various cellular processes (e.g., DNA replication, neuronal survival, glial cell proliferation), non-cholinergic biochemical pathways (e.g., serotoninergic synaptic functions, the adenylate cyclase system), and causes various behavioral abnormalities (e.g., locomotor skills, cognitive performance) [74], [75], [76], [77], [78], [79], [80], [81] and [82]. Interesting observations have also been provided by in vitro studies. For example, various OPs have been shown to inhibit astroglial cell proliferation [83] and [84] and to cause neuronal apoptotic death [85]. These effects were seen, however, at relatively high OP concentrations. On the other hand, chlorpyrifos and its oxon were found to inhibit axonal outgrowth and to increase dendritic outgrowth in rat sympathetic neurons derived from superior cervical ganglia, at very low concentrations, below those required to inhibit AChE catalytic activity [86].

These findings, together with results of biomonitoring studies that indicate exposure of children, particularly in inner cities and in farming communities, to OPs [87] and [88], have led to regulatory restrictions on the use of certain OPs, and to heightened concern for their potential neurotoxic effects in children [89], [90] and [91]. Furthermore, specific guidelines for developmental neurotoxicity testing have been implemented [92]. Given the right importance attributed to children‘s health in our society, there is the need for a better understanding of the potential developmental neurotoxicity of OPs. If effects are secondary to AChE inhibition, careful consideration of this endpoint must be undertaken to assure that current exposure limits guarantee the safety of children. However, studies should also focus on possible effects of the parent-compound, unrelated to AChE inhibition, and on possible noncholinergic targets (see also Section 3.6).

3.4. Common mechanism of action

Human health risk assessments for pesticides, including OPs, have been routinely performed using the reference dose (RfD) or acceptable daily intake (ADI) approaches, which define an acceptable level of human exposure to compounds, that would be without appreciable risks of deleterious effects during a lifetime of exposure to the general population, including sensitive subgroups. No-observed-adverse-effect-levels (NOAELs) are derived from subchronic or chronic animal studies identifying the most sensitive adverse effect; division of the NOAEL by uncertainty factors (generally based on interspecies extrapolation and intraspecies variability) allow the determination of the RfD (or ADI). Even though an individual may be exposed to more than one OP at any given time (e.g., through consumption of multiple residues on foods), risk characterization has been based on toxicity of, and exposure to, individual chemicals [93]. With the enactment by the US Congress of the Food Quality Protection Act (FQPA) in 1996, the US Environmental Protection Agency (US EPA) is now required to conduct combined risk assessments for pesticides showing a “common mechanism of toxicity” [65]. In essence, if two or more pesticides are considered to act through a common mechanism, then cumulative effects of co-exposure would have to be considered in the evaluation and setting of tolerance levels.

In case of OPs, all indeed act by phosphorylating AChE and eliciting a spectrum of cholinergic effects [94]. The US EPA has thus developed a revised cumulative risk assessment for OPs, based on the potency in inhibiting AChE and on the assumption of dose additivity [95]. A decision to perform such combined risk assessments on all OPs based on the interaction with AChE as the common mechanism of action, and hence, of toxicity, may potentially restrict the use of some OPs, particularly when multiple residues of OPs are found in certain foods and when aggregate exposures from nondietary routes are also considered, as required by FQPA [93]. Determining whether all OPs have the same mechanism of toxicity or if sufficient differences in toxic mechanisms exist to allow subclassification, is thus a current topic of much debate. A few years ago, an ILSI working group formulated six hypotheses for possible subclassifcation of OPs, based on differences in OP metabolism, distribution and molecular targets [94]. However, all hypotheses were rejected, and the working group conducted that all “OPs should be considered to act as a common mechanism of toxicity if they inhibit AChE by phosphorylation and elicit any spectrum of cholinergic effects” [94]. Nevertheless, it is being argued that available information are not sufficient to allow a meaningful cumulative risk assessment for OPs. Some of the arguments are as follows: 1) OP exposures causing similar levels of AChE inhibition may induce a different spectrum of toxic signs; 2) additivity cannot be always assumed, as one OP may modify (increase or decrease) the toxicity of another OP; 3) non-cholinergic targets (i.e., targets other than AChE) exist that may be involved in the differential toxicity of OPs and may modify cholinergic toxicity [57], [93] and [95]. The latter argument is of particular interest because it may hinge on various aspects discussed in this section, such as chronic low toxicity and developmental toxicity of OPs, and will be discussed more in detail below (see Section 3.6). Yet, with regard to a common mechanism of action for OPs, the question appears to be still open.

3.5. OP-induced delayed neurotoxicity

In a previous section, NTE has been described as the serine esterase representing the primary target for OPIDP. NTE is defined as the phenylvalerate hydrolyzing activity that is resistant to paraoxon but is inhibited by mipafox, a neuropathic OP [14]. This is a “working definition” that has proven useful for research on OPIDP over the years [11], but does not shed light on the nature and physiological action of the enzyme, nor on its clear role in OPIDP, aside of that of being the target for initiators. Molecular research in the past few years and the use of transgenic animal models have provided some novel and at times unexpected information.

NTE is a member of a novel protein family represented in organisms from bacteria to man; of its 1327 amino acids, 200 near the C-terminal are highly conserved, and the active site serine (S966) lies within this region [96]. Atkins and Glynn expressed an active site region corresponding to residues 727–1216 in E. coli and referred it as NEST (NTE-esterase domain) [97]. This made it possible to study substrate specificity, which showed preferential hydrolysis of lipids [98]. A more recent study showed that NTE in mammalian cells and its homologue in yeast degrade phosphatidylcholine to glycerophosphocholine, thereby substantiating a primary role in lipid homeostasis [99]. Quistad et al. provided evidence that NTE may be a lysophospholipase (LysoPLA), which hydrolyzes lysolecithin, a major membrane phospholipid which has demyelinating properties [100]; neuropathic compounds were found to inhibit NTE and NTE-LysoPLA to the same extent, both in vitro and in vivo [100] and [101].

The primary sequence of human NTE has a 41% identity with the Swiss Cheese Protein (SWS) in neurons of Drosophila. In the developing nervous system of Drosophila SWS is possibly involved in cell–cell communication between neurons and glia, but in SWS mutants, aberrant cell–cell interactions lead to apoptotic neuronal and glial death, with extensive vacuolation in the brain (hence the name) [102]. Deletion of NTE gene was found to be lethal in mice at an early embryonic age [103] and [104]. Though embryonic lethality of NTE was indicated to be due to placental failure and impaired vasculogenesis, rather than loss of NTE expression by neurons [104], this nevertheless precluded the study of the role of NTE in the adult brain. A conditional mutant NTE strain where NTE was deleted in neuronal tissue did not show embryolethality and provided viable offspring [105]. These animals (Nes-cre: NTE^fl/fl) had less than 10% of NTE activity, but no NTE protein could be detected by Western blot. AChE activity was normal. When the animal brains were examined at 3–4 months of age, significant vacuolization and a dramatic redistribution of the rough endoplasmic reticulum was found in the hippocampus and the thalamus. Loss of Purkinje cells in the cerebellum was also present, with parallel motor deficits in a rotarod test [105].

Additional studies in Drosophila added further interesting information. As said, SWS mutants show vacuolization and degeneration of neurites. This could be reversed by murine NTE, indicating that the mouse protein (and most likely the human NTE protein, which is 96% identical to the murine one) is a true functional ortholog of fly SWS [106]. In Drosophila, SWS is expressed in neurons and to a minor extent in glia, and is localized in the endoplasmic reticulum; a similar distribution had been found for NTE in mouse brain, though no NTE had been detected in glia [106] and [107]. Phosphatidylcholine levels were increased in SWS mutants, substantiating the role of SWS/NTE in its hydrolysis [99], and in the regulation of normal lipid composition in the CNS [106].

While loss of both NTE alleles resulted in embryolethality, heterozygous (Nte^+/?) mice were viable and fertile [103]. Nte^+/? mice had 40% less brain NTE activity than wild-type mice, but equal brain AChE activity. Nte^+/? mice also displayed a phenotype of hyperactivity, which is not directly relevant to OPIDP, and were more sensitive to the acute toxicity of ethyl octophosphoro-fluoridate (EOPF), an OPIDP-causing OP [103]. Following administration of a low dose of EOPF, Nte^+/? mice showed a further increased locomotor activity, but NTE activity was not quantified in these animals [103].

Do these studies contribute to our understanding of OPIDP? Unfortunately, so far only to a very limited extent. Clearly, NTE seems to play a role in membrane lipid metabolism, and may be involved in intra-neuronal membrane trafficking and lipid homeostasis. Whether its lysoPLA activity may be involved in OPIDP remains to be clarified, as this enzymatic activity is also affected by chemicals that do not cause OPIDP. Accumulated lysolecithin causes demyelination of neuronal sheaths, often accompanied by neuronal lesions [100], while changes in axonal morphology seem to be the early morphological events in OPDIP, with secondary attenuation of the myelin sheath [12]. Studies in genetically modified mice have provided some interesting insights in the physiological role of NTE. NTE appears to be required for normal blood vessel and placental development [103] and [104], and absence of brain NTE results in neuronal degeneration and loss of endoplasmic reticulum in various brain areas [105], two effects not directly related to OPIDP. Furthermore, mice, though an excellent tool for transgenic research, are resistant to OPIDP [18]. Thus, the most crucial issues in the mechanisms of OPIDP development and progression still remain obscure [13].

3.6. Additional OP targets

The possibility that, in addition to the previously discussed AChE, NTE, BChE and carboxylesterase, OPs may act on the other targets, and the relevance that such interactions may have in mediating some of the effects of OPs, are another matter of continuing debate. For example, it has been pointed out that the pattern of symptoms is different with different OPs, and cannot be explained by inhibition of AChE alone [57], [93] and [107a]. This argument would have relevance for the discussion of common mechanism of action of OPs and the proposed cumulative risk assessment (see Section 3.4). The debate on the possible CNS effects of long-term chronic exposure also calls for the involvement of additional targets (see Section 3.1). Furthermore, developmental neurotoxicity has been reported at OP doses that cause minimal or no AChE inhibition, suggesting again the possibility that OPs may affect other biological systems (see Section 3.3).

Over the years, OPs have been shown to affect hundreds of enzymes, as well as receptors and signal transduction systems. Thus, a first important issue is to compare the relative potency of OPs toward these targets and toward AChE. It is also important to determine whether the observed effect or interaction also occur with the parent compound, whose concentrations in the body may be higher than that of the oxon. Verification of the findings in vivo would also be relevant.

In addition to AChE, OPs have been shown to interact or affect, other components of the cholinergic system. Several OPs were found to bind to nicotinic receptors, however, only at high concentrations (100 μM; [108]). OPs can also interact at lower concentration (nanomolar range) with muscarinic receptors, particularly the M₂ subtype [109] and [110]; and references in 111], where they appear to act as agonists. In vivo, OPs are known to cause down-regulation of muscarinic receptors, and this is believed to be an indirect effect, due to accumulated acetylcholine [33]. However, contribution of a direct OP-muscarinic receptor interaction cannot be ruled out. Other pre- and post-synaptic components of the cholinergic system have also been reported to be affected by OPs. These include choline acetyltransferase, high affinity choline uptake, the vesicular acetylcholine transporter, and different kinases involved in signal-transduction. Such effects have been seen in vitro, often at rather high concentrations [112], as well as in vivo, but it is not clear whether in the latter case, they represent compensatory mechanisms to cholinergic overstimulation.

Enzymes involved in the metabolism of peptides have been reported to be affected by OPs. For example, acylpeptide hydrolase (APH) is inhibited by various oxons at concentrations lower than those required to inhibit AChE; inhibition was also observed after in vivo exposure to OPs [113]. APH is responsible for the removal of N-acetylated aminoacids from the N-terminal of short peptides, such as α-melanocyte-stimulating hormone or β-endorphin. Indirect evidence for inhibition of peptidases involved in the metabolism of enkephalins was reported in a comparative analysis of DFP- and physostigmine-induced antinociception in mice [114] and [115]. DFP, but not physostigmine, significantly increased met-enkephalin levels in the striatum. Both compounds caused antinociception, however, only that induced by DFP was antagonized by naloxone. These in vivo effects, however, were seen of doses that caused significant AChE inhibition.

Another system that has been recently shown to be targeted by OPs is the cannabinoid system. The endocannabinoid anandamide binds to the cannabinoid receptor (CB1) in brain, which is also the target for Δ9-tetrahydocannabinol, the principal psychoactive ingredient of marijuana [116]. Both anandamide and the endogenous sleep-induced agent oleamide are hydrolyzed by fatty acid amide hydrolase (FAAH). Various OPs are potent inhibitors of FAAH [117], and may increase CB1-mediated responses, such as hypomotility, analysis, catalepsy and hypothermia [118]. Of several OPs tested (both oxons and parent compounds) only chlorpyrifos oxon was a potent inhibitor of CB1 binding in vitro [119]. However, in vivo administration of symptomatic doses only caused a 25% inhibition [119]. All OPs tested were more potent toward FAAH then CB1, but the relevance of these targets in OP toxicity is unclear [111].

Overall, the possibility that OPs may act on targets other than AChE is tempting and interesting. However, research so far, even in the area of developmental neurotoxicity (see Section 3.3) has not provided sufficient evidence that such targets may contribute to adverse health effects of OPs.

4. Conclusion

The use of OPs as insecticides in the agricultural and urban settings is still high and is expected to remain so, at least in the near future. While other classes of insecticides are gaining market share (e.g., pyrethroids) and new classes have been developed (e.g., neonicotinoids), the efficacy of OPs, their relatively low cost and their lack of bioaccumulation in the ecosystems, would support this prediction. Yet OPs display relatively limited selectivity (one exception may be malathion) between insects and non target species, including humans. As such, concerns on their potential adverse effects in human populations will continue. The issues discussed in the review still represent real-life problems, with clinical, societal and legal ramifications. Continuing research in all these areas, and others not mentioned, is welcome and warranted.

Acknowledgements

Research by the author is supported by grants from the National Institute of Environment Health (ES04696, ES07033, ES09601/EPA-R826886). Views expressed in this paper are solely of the author and not of the funding agencies.

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