Genome-Wide Gene Expression Analysis in Response to Organophosphorus Pesticide Chlorpyrifos and Diazinon in C. elegans

Organophosphorus pesticides (OPs) were originally designed to affect the nervous system by inhibiting the enzyme acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Over the past years evidence is mounting that these compounds affect many other processes. Little is known, however, about gene expression responses against OPs in the nematode Caenorhabditis elegans. This is surprising because C. elegans is extensively used as a model species in toxicity studies. To address this question we performed a microarray study in C. elegans which was exposed for 72 hrs to two widely used Ops, chlorpyrifos and diazinon, and a low dose mixture of these two compounds. Our analysis revealed transcriptional responses related to detoxification, stress, innate immunity, and transport and metabolism of lipids in all treatments. We found that for both compounds as well as in the mixture, these processes were regulated by different gene transcripts. Our results illustrate intense, and unexpected crosstalk between gene pathways in response to chlorpyrifos and diazinon in C. elegans

The epithelial cholinergic system of the airways

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types—ciliated, basal and secretory—are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs

REDUCED ACETYLCHOLINE RECEPTOR DENSITY, MORPHOLOGICAL REMODELING, AND BUTYRYLCHOLINESTERASE ACTIVITY CAN SUSTAIN MUSCLE FUNCTION IN ACETYLCHOLINESTERASE KNOCKOUT MICE

The vertebrate neuromuscular junction is designed for rapid transmission of excitatory signals for initiation of muscle contraction.5 Among the features responsible for the high throughput of this synapse are the close proximity of the presynaptic and postsynaptic membranes,10 the direct coupling of acetylcholine (ACh) binding to the opening of the ion channel associated with the nicotinic acetylcholine receptor (nAChR),27 the brief open time of this channel,21,27 and the presence of cholinesterase (ChE) for hydrolysis of ACh.21,30 At the endplate, there are two distinct ChEs for transmitter hydrolysis: acetylcholinesterase (EC 3.1.1.7, AChE) and butyrylcholinesterase (EC 3.1.1.8, BChE).33 Both enzymes can exist in a multisubunit, collagen-tailed form with selective localization at the endplate basallamina.33 Because of its superior catalytic activity for ACh hydrolysis, AChE is the dominant enzyme, whereas the role of BChE is generally evident only after AChE is inhibited.3,4 Inhibition of ChE results in a progressive accumulation of ACh, especially during periods of repetitive stimulation, leading to desensitization of nAChRs and consequent muscle weakness.12,17 Under this condition, transmitter persists beyond its normal lifetime and is slowly removed from the endplate region by diffusion.21,30 Diffusion is impeded in part by morphological barriers, such as the apposition of the nerve terminal to the postjunctional membrane,5,10 and by the high density of postjunctional nAChRs.21,22,25 If ChE is inhibited pharmacologically or removed by collagenase treatment, repeated binding to nAChR makes diffusional loss of ACh slow and inefficient.21,22,30 The influence of nAChRs on retention of transmitter was termed “buffered diffusion” by Katz and Miledi21 and accounts for findings that elimination of ACh is considerably slower than that expected for free diffusion. 30 Inhibitors of ChE are highly toxic, producing incapacitation and death within minutes.28 The cause of death is complex, involving loss of central respiratory drive,6,29 bronchospasm,1,2 and the inability of the diaphragm muscle to sustain tetanic tension. 19 Because most ChE inhibitors show little selectivity between AChE and BChE, and may have direct actions unrelated to ChE inhibition, it is difficult to establish the role of AChE activity in neuromuscular transmission. To overcome this difficulty, we studied twitch and tetanic tensions in diaphragm muscles from AChE knockout (AChE-/-) mice that fail to express AChE but do contain normal levels of BChE.7,24,36 Stimulation of the phrenic nerve in isolated diaphragm preparations from AChE-/- mice revealed large single twitches and sustained tetanic tensions at 70 and 100 Hz. These findings suggest that, over a limited frequency range, diaphragm muscles from AChE-/- mice are able to compensate for the loss of AChE activity. An understanding of these adaptive mechanisms is expected to provide insight on protection strategies that may be effective against the toxic actions of ChE inhibitors such as the highly lethal nerve agents

REDUCED ACETYLCHOLINE RECEPTOR DENSITY, MORPHOLOGICAL REMODELING, AND BUTYRYLCHOLINESTERASE ACTIVITY CAN SUSTAIN MUSCLE FUNCTION IN ACETYLCHOLINESTERASE KNOCKOUT MICE

The vertebrate neuromuscular junction is designed for rapid transmission of excitatory signals for initiation of muscle contraction.5 Among the features responsible for the high throughput of this synapse are the close proximity of the presynaptic and postsynaptic membranes,10 the direct coupling of acetylcholine (ACh) binding to the opening of the ion channel associated with the nicotinic acetylcholine receptor (nAChR),27 the brief open time of this channel,21,27 and the presence of cholinesterase (ChE) for hydrolysis of ACh.21,30 At the endplate, there are two distinct ChEs for transmitter hydrolysis: acetylcholinesterase (EC 3.1.1.7, AChE) and butyrylcholinesterase (EC 3.1.1.8, BChE).33 Both enzymes can exist in a multisubunit, collagen-tailed form with selective localization at the endplate basallamina.33 Because of its superior catalytic activity for ACh hydrolysis, AChE is the dominant enzyme, whereas the role of BChE is generally evident only after AChE is inhibited.3,4 Inhibition of ChE results in a progressive accumulation of ACh, especially during periods of repetitive stimulation, leading to desensitization of nAChRs and consequent muscle weakness.12,17 Under this condition, transmitter persists beyond its normal lifetime and is slowly removed from the endplate region by diffusion.21,30 Diffusion is impeded in part by morphological barriers, such as the apposition of the nerve terminal to the postjunctional membrane,5,10 and by the high density of postjunctional nAChRs.21,22,25 If ChE is inhibited pharmacologically or removed by collagenase treatment, repeated binding to nAChR makes diffusional loss of ACh slow and inefficient.21,22,30 The influence of nAChRs on retention of transmitter was termed “buffered diffusion” by Katz and Miledi21 and accounts for findings that elimination of ACh is considerably slower than that expected for free diffusion. 30 Inhibitors of ChE are highly toxic, producing incapacitation and death within minutes.28 The cause of death is complex, involving loss of central respiratory drive,6,29 bronchospasm,1,2 and the inability of the diaphragm muscle to sustain tetanic tension. 19 Because most ChE inhibitors show little selectivity between AChE and BChE, and may have direct actions unrelated to ChE inhibition, it is difficult to establish the role of AChE activity in neuromuscular transmission. To overcome this difficulty, we studied twitch and tetanic tensions in diaphragm muscles from AChE knockout (AChE-/-) mice that fail to express AChE but do contain normal levels of BChE.7,24,36 Stimulation of the phrenic nerve in isolated diaphragm preparations from AChE-/- mice revealed large single twitches and sustained tetanic tensions at 70 and 100 Hz. These findings suggest that, over a limited frequency range, diaphragm muscles from AChE-/- mice are able to compensate for the loss of AChE activity. An understanding of these adaptive mechanisms is expected to provide insight on protection strategies that may be effective against the toxic actions of ChE inhibitors such as the highly lethal nerve agents