Digital Object Identifier (DOT) l0.1007/s002040000111
LETTER TO THE EDITOR
U. MuBhoff, M. Madeja, N. Binding, U. Witting, E.-J. Speckmann.
2-Phenoxyethanol: a neurotoxicant? Reply.
Arch Toxicol (2000) 74: 284-287
Received: 1 March 2000 / Accepted: 1 March 2000 /Published online: 17 May 2000 © Springer-Verlag 2000
U. Mubhoff, M. Madeja, E.-J. Spcckmann, Institut für Physiologie, Robert-Koch-Strasse 27a, 48149 Munster. Germany
N. Binding U. Witting
Institut für Arbeitsmedizin, Robert-Koch-Strasse 51, 48149 Munster, Germany
We tested the possible interactions of 17 different glycol ether compounds with two glutamate subreceptors and published the data recently (Mulbhoff et al. 1999). The glutamate receptors sensitive to the agonists N-methyl-D-aspartate (NMDA) and kainate were heterologously expressed in Xenopus oocytes by injection of rat brain RNA. The effects of the glycol ether compounds on the function of these receptors were examined with the voltage clamp technique. The investigations revealed that ethylene glycol monophenyl ether (2-phenoxyethanol) reduced NMDA-induced ion currents, while the kainate reaction was not affected by this substance. The threshold concentration of 2-phenoxyethanol was less than 10 umol/1; the inhibitory concentration for 50% of the group (IC50) was 362 umol/l. We suggest that this effect of 2-phenoxyethanol on the NMDA receptor possibly involves a neurotoxic potential of this substance.
In a letter to the editor, Schmuck, Steffens, and Bromhard criticized the in vitro model used as well as the interpretation of the data which we presented in our discussion. The authors stated that an "inhibition of the NMDA receptor does not lead necessarily to an impairment of memory function nor to other symptoms". Briefly, they maintain this for two reasons: inhibitors of NMDA receptors, such as MK-801, show neuroprotective effects in epilepsy and stroke, and long-term potentiation is not exclusively regulated by the NMDA receptor. Furthermore, they stated that the inhibitory effect of 2-phenoxycihanol on NMDA-induced membrane currents is probably caused by an unspecific action of this substance. We would like to comment on this.
1. NMDA receptors play an important role in brain function and in the pathogenesis of several brain dysfunctions such as epilepsy and stroke. For this reason, several NMDA receptor antagonists have been developed and tested for protection from these brain dysfunctions. For example, MK-801 (dizocilpine) is a highly potent, selective, and noncompetitive NMDA receptor antagonist that acts by binding to a site located within the NMDA-associated ion channel, thus preventing Ca2+ influx. The substance is an effective anti-ischemic agent in several animal models and has been used to develop an in vitro model of NMDA-receptor-dependent apoptotic neurodegenerative disease in developing rat brains. Furthermore, it has been used to develop an in vitro model of NMDA-receptor-specific Ca2+ entry-induced epilepsy.
However, this and other noncompetitive and competitive NMDA receptor antagonists produce serious side effects in the tested animals, including altered sensory processing, disruption of learning and memory, and stereotypes such as motor impairment and head wearing (Fraser 1996; Hargreaves and Cain 1992; Löscher and Hönack 1991; Meldrum 1995; Rogawski 1992). Moreover, a particularly troubling action of MK-801 is that it causes neuropathological changes (e.g., vacuolization) in neurons of certain areas of the brain (Hargreaves et al. 1994; Leeson and Iversen 1994; Olney et al. 1989, 1991). Furthermore, MK-801 and other NMDA antagonists elicit an enhancement of autonomic nervous system function which leads to elevated heart rate and blood pressure at neuroprotective doses, a potentially serious complication in the clinical use of these compounds in stroke patients (Leeson and Iversen 1994). In humans, NMDA antagonists cause disturbances (eg., sedation, ataxia, stupor, dysarthria, confusion, hallucinations, agitation, reduced pain perception, and auditory, visual, proprioceptive, and attentional disturbances) that arc consistent with the anatomical distribution and physiological functions of NMDA receptors (for review, see Cain 1997).
In summary, NMDA antagonists produce a variety of adverse neurobehavioral and neurotoxicological effects, something not quite unexpected when considering the involvement of NMDA receptors in so many critical brain functions. Because of these profound side effects, most of the NMDA antagonists are unlikely to become clinically viable, and evaluation of MK-80l in human subjects has been terminated (Hudspith 1997; Leeson and Iversen 1994; Rogawski 1992). Taking these findings into account, we feel it is by no means an exaggeration to assume that 2-phenoxyethanol, which caused similar effects on NMDA receptor function as the above-mentioned substances, has a neurotoxic potential.
2. Long-term potentiation (LTP) and long-term depression (LTD) are currently the best available cellular models for learning and memory in the mammalian brain (Larkman and Jack 1995; Dudek and Bear 1992). LTP is defined as a long-term increase in synaptic efficacy induced by the high-frequency stimulation of excitatory afferent pathways. It is widely agreed that LTP induction is mediated largely, but not necessarily exclusively (Huber et al. 1995; Morgan and Teyler 1999), by the activation of NMDA receptors (Larkman and Jack 1995). Gene knockout experiments have confirmed the importance of this receptor in LTP (Sakimura Ct al 1995).
Therefore, it is clear that inhibition of NMDA receptor function has the potential to block the induction of LTP. In fact, application of NMDA antagonists, like MK-801 or ethanol, significantly reduce or completely block LTP and impair cognitive functions (see, for example, Bliss and Collingridge 1993; Collingridge and Bliss 1995; Hudspith 1997; Morgan and Teyler 1999). From this point of view, it is again not an exaggeration to assume that 2-phenoxyethanol, which caused similar effects on NMDA receptor function as the above-mentioned substances, can also exert profound effects on LTP/LTD and thus on central cognitive functions.
3. Schmuck, Steffens, and Bromhard propose a rather nonspecific receptor interaction of the solvent 2-phenoxyethanol with the NMDA receptor. They argued that solvents like ethanol exert their unspecific inhibitory effects on the transmitter receptors for NMDA, gammaaminobutyric acid (GABA), acetylcholine (mACh) via an unspecific alteration of the neuronal membrane.
In fact, there is extensive evidence that ethanol interacts with a variety of neurotransmitters. The major actions involve enhancement (!) of GABA at the GA-BAA receptor and blockade of the NMDA receptor (for reviews, see Crews et al. 1996; Feingold et al. 1998, Tsai and Coyle 1998). In line with a specific action of ethanol is the fact that ethanol increases GABAA-mediated inhibition, but this does not occur in all brain regions, for all cell types in the same region, nor at all GABAA receptor sites on the same neuron, nor in the same brain region across species. The molecular basis for the selectivity of the action of ethanol on GABAA receptors has been intensively studied. Overall it is the appropriate combination of GABAA receptor subunits (benzodiazepine receptor b2 subunit, and alternative splice variants of the Y2 subunit) that appears to be important for ethanol enhancing the action of GABA (Brozowski et al. 1995; Criswell et al. 1993; Whatley et al. 1995). Chimeric GABAA receptor constructs have identified a region of 45 amino-acid residues necessary for the enhancement of the function of this receptor by ethanol. Two specific amino-acid residues in transmembrane domain 2 and 3 of this region are critical for the effects of ethanol (Mihic et al. 1997).
As is the case for GABAA receptors, ethanol affects NMDA receptors depending on the subunit combination (for reviews see Crews Ct al. 1996; Feingold et al. 1998; Tsai et al. 1998). A differential sensitivity of recombinant NMDA receptor subunit combinations to ethanol has been reported, which may contribute to the differences in ethanol sensitivity observed in different types of neurons in vivo and in vitro (Masood et al. 1994). Ethanol inhibition of recombinant NMDA channels was mediated, in part, by action on the NMDAR2B subunit (Blevins et al. 1995; Chu et al. 1995; Popp Ct al. 1996). Changes in the redox state of the NMDA receptor may also affect the sensitivity of the receptor to the inhibitory actions of ethanol in the hippocampus (Woodward 1994). For these reasons, it seems to be rather unlikely that general alterations of the neuronal membranes are responsible for the effects of ethanol on GABA and glutamate receptors. It is more likely that ethanol causes effects by binding to specific sites of the receptor complexes. The blockade of the NMDA receptor may be crucial to the neurotoxic effects of ethanol. Thus, acute dysfunction of the NMDA receptor may account for many of the symptoms of intoxication including euphoria and blackouts as well as impairment of learning and memory (Tsai et al. 1998). Since 2-phenoxyethanol selectively inhibits the NMDA receptor (in our experiments kainate responses were not affected), it is likely that this compound, and probably also ethanol, interacts in a specific way with the NMDA receptor and not via a nonspecific interaction with the surrounding membrane. One can assume that the dysfunction of the NMDA receptor by 2-phenoxyethanol may exert similar neurotoxic effects in vivo.
In this context, it should be mentioned that Xenopus oocytes are well known to be excellently suited to explore the function of neurotransmitter receptors, their molecular composition, and drug actions on these receptors. We chose the oocyte expression system for these studies because we were primarily interested in a direct assessment of acute effects on the glutamate receptors. Xenopus oocytes are capable of expressing foreign receptors with pharmacological properties very similar, if not identical, to those in brain. Thus, the single-cell system permits a direct study of ligand-operated ion channels without confounding factors, especially transmitter release or uptake from neighboring cells. In many of the above cited literature studies, oocytes were used to investigate the effects of antagonists (for example. MK80l) and drugs (for example, ethanol) on the ligandoperated ion channels.
A short remark follows, addressed to the criticism of Schmuck. Steffens and Bromhard concerning the literrature mentioned in our publication.
1. We cited the report of Morton (1990) in which neurotoxic effects in three women were ascribed to probable 2-phenoxyethanol poisoning. Schmuck, Steffens, and Bromhard criticized this report in many aspects and mentioned "serious doubts about whether there is a causal relationship between exposure to 2-phenoxyethanol and these effects". Their criticism is mainly based on "personal discussions with Prof. Morton". In our reports, we accepted and cited only published data in scientific journals which have been reviewed by a referee system. In scientific journals, "personal communications" are usually cited only in exceptional cases and should be documented by a written statement from the investigator concerned. We generally avoid personal communications or other inaccessible information, especially in such cases when the work of others (in this case that of Morton himself and our work) are criticized. For these reasons, we cannot accept the interpretation and criticism of this article by Schmuck, Steffens, and Bromhard, and refer to the published data of Morton.
2. We cited a work by Ahmed and co-workers in which they showed that ethylene glycol monomethyl ether and/or its metabolites could rapidly cross the blood-brain barrier at an early time period after treatment. Unfortunately, we inadvertently quoted the wrong article of this group. They published the above results in the following report: Ahmed et al. (1994).
In summary, we found an antagonistic effect of 2-phenoxyethanol on the NMDA responses in voltage-clamp experiments with the Xenopus oocyte expression system. Since most of the NMDA antagonists exert profound neurobehavioural and neurotoxic effects, we discussed the possibility that 2-phenoxyethanol also possesses a neurotoxic potential, a conclusion which certainly is fully justified when considering all our and other published data.
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