The nematode Caenorhabditis elegans as a model of organophosphate-induced mammalian neurotoxicity

Abstract

Fifteen organic phosphate pesticides were tested by computer tracking for their acute behavioral toxicity with the nematode Caenorhabditis elegans. Thirteen of these 15 chemicals are used as insecticides and are anticholesterase agents. The other two chemicals are used as herbicides. EC50 values for each chemical were compared to the corresponding LD50 acute lethality value in rats and mice. Order of toxicity was found to be significantly correlated in comparisons of C. elegans to both rats and mice. Mechanistic investigations were conducted by assaying 8 of the 15 chemicals for anticholinesterase activity in C. elegans. Significant cholinesterase inhibition was confirmed for five chemicals that had displayed high behavioral toxicity, while three chemicals of low behavioral toxicity showed no significant decrease in cholinesterase activity. Toxicity for two chemicals that do not inhibit cholinesterase in mammals was linked to pH effects. Detailed comparison of individual chemicals and metabolic issues are discussed. These results have positive implications for the use of C. elegans as a mammalian neurological model and support the use of C. elegans in early rounds of chemical toxicity screening.

Introduction

The study of biological questions often proceeds through the investigation of model systems. Of those organisms that have become established as biological models, few have the scope and breadth of the nematode Caenorhabditis elegans. C. elegans, a nonparasitic bacterial feeder that lives in soil interstitia, has served as a model in neurobiology, developmental biology, and genetics. The potential of C. elegans as a model organism was recognized by Brenner (1974) early in the process of its initial characterization. Several unique advancements in the study of C. elegans biology over the past 30 years have brought Brenner's insight to fruition. In 1983, Sulston et al. completed a milestone in developmental biology by tracing the lineage of each cell in the adult hermaphrodite from differentiation to death. A few years later, White et al. (1986) published a complete map of the C. elegans nervous system reconstructed from serial section electron micrographs. More recently, genetic distinction has come to C. elegans by being the first multicellular organism to have its genome completely sequenced (C. elegans Sequencing Consortium, 1998). The enormous body of C. elegans research that is highlighted by these accomplishments arguably makes C. elegans the most thoroughly characterized organism on Earth.

C. elegans is one of the simplest organisms with a centralized nervous system. Despite the phylogenetic distance between nematodes and vertebrates, they share many similarities with respect to neural physiology. The genomic sequencing of C. elegans has brought to light an unexpected level of conservation with vertebrates. Homology and conserved gene structure have been identified in genes responsible for many neural components including ion channels, neurotransmitter biosynthetic enzymes, synaptic release mechanisms, neurotransmitter receptors, and second messenger systems (Bargmann, 1998). Strong evidence for neurotransmitter-mediated transduction of the synapse in C. elegans has been demonstrated for acetylcholine, GABA, serotonin, dopamine, glutamate, and several neurogenic peptides (Rand and Nonet, 1997).

There are several logistical considerations that make C. elegans appealing as a neurological model. With a total of 302 neurons in the adult hermaphrodite, C. elegans offers simplified anatomical complexity compared to higher organisms without sacrificing its genetic and physiological applicability. The progression of each neural cell has been characterized (Sulston et al., 1983), and the almost invariant arrangement of its 5000 chemical synapses and 2000 neuromuscular junctions is known (White et al., 1986). This relatively simple nervous system supports a few basic behaviors that have been studied quantitatively (Sulston and Hodgkin, 1988).

As in higher animals, acetylcholine (ACh) is the primary excitatory neurotransmitter involved in C. elegans motor function (Rand and Nonet, 1997). Also conserved is the synaptic degradation of ACh by acetylcholinesterase (AChE) enzymes. While vertebrates have a single AChE gene whose product can undergo alternative splicing post-transcriptionally to yield different isoforms, C. elegans has four separate genes yielding four AChE enzymes (Combes et al., 2001). Two of these enzymes, ACE-1 and ACE-2, account for roughly 95% of all AChE activity in C. elegans. Though they share only 35% identity, ACE-1 and ACE-2 display a high degree of functional redundancy (Combes et al., 2001). Johnson and Russell (1983) found that dual mutations nullifying the product of both genes were required to produce a phenotype readily distinguishable from the wild type. ACE-3 and ACE-4 are minor forms of AChE in C. elegans accounting for approximately 5% and 0.1% of wild-type activity, respectively (Combes et al., 2001). ACE-3 exhibits enough activity for ace-1, ace-2 double mutants to be viable, but movement and development are severely degraded.

C. elegans has been used in testing environmental contaminants known to have neural toxic effects. By developing a neurotoxicity index from lethality data, Williams and Dusenbery (1990a) concluded that lead (Pb), mercury (Hg), and two organophosphate pesticides (malathion and dichlorvos) displayed concentration–response dynamics consistent with neurotoxicity although copper (Cu) did not. The idea that movement behavior may be preferentially sensitive to neurotoxicants has led several researchers to use locomotion in the examination of chemicals known to be neurotoxic. Anderson et al. (2001) demonstrated that movement in C. elegans was more sensitive to Pb than to Cu following 4-h exposures, although this difference in sensitivity was masked after exposures lasting 24 h.

Efforts have also been made to evaluate C. elegans as a model for mammalian toxicity. Williams and Dusenbery (1988) found that acute lethality values in C. elegans exposed to eight heavy metallic salts predicted overall mammalian acute lethality as consistently as tests in rats and mice individually. Williams et al. (2000) conducted blind tests on five gadolinium-based compounds used in magnetic resonance imaging measuring movement behavior as the quantified endpoint. These tests correctly ordered the toxicity for four out of the five compounds compared to minimum lethal doses for the same compounds in mice. Anderson et al. (in press) found behavioral toxicity testing following 4-h exposures correctly ordered five compounds across three chemical classes when referenced to toxicity in rats and mice.

The purpose of this investigation was to explore the use of C. elegans as a model for mammalian neurotoxicity by testing its response to organophosphorus compounds. We looked at this question in two ways. Our first hypothesis was that the relative order of toxicity for a group of organic phosphate substances tested in C. elegans using behavioral methods would bear significant similarity to their toxicity order in mammals. We also hypothesized that for the organophosphate (OP) insecticides (13 of the 15 chemicals tested), the mechanism by which they exert their toxic effects in C. elegans would be the same as for mammals (cholinesterase inhibition).