Chronic toxicity of ibuprofen to Daphnia magna: Effects on life history traits and population dynamics

The non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) is a widely used pharmaceutical that can be found in several freshwater ecosystems. Acute toxicity studies with Daphnia magna suggest that the 48 h EC50 (immobilisation) is 10-100 mg IB l(-1). However, there are currently no chronic IB toxicity dataon arthropod populations, and the aquatic life impacts of such analgesic drugs are still undefined. We performed a 14-day exposure of D. magna to IB as a model compound (concentration range: 0, 20, 40 and 80 mg IB l(-1)) measuring chronic effects on life history traits and population performance. Population growth rate was significantly reduced at all IB concentrations, although survival was only affected at 80 mg IB l(-1). Reproduction, however, was affected at lower concentrations of IB (14-day EC50 of 13.4 mg IB l(-1)), and was completely inhibited at the highest test concentration. The results from this study indicate that the long-term crustacean population consequences of a chronic IB exposure at environmentally realistic concentrations (ng l(-1) to mu g l(-1)) would most likely be of minor importance. We discuss our results in relation to recent genomic studies, which suggest that the potential mechanism of toxicity in Daphnia is similar to the mode of action in mammals, where IB inhibits eicosanoid biosynthesis. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous alpha,beta-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

A mesoscopic modelling approach to anaesthetic action on brain electrical activity

Relating the measurable, large scale, effects of anaesthetic agents to their molecular and cellular targets of action is necessary to better understand the principles by which they affect behavior, as well as enabling the design and evaluation of more effective agents and the better clinical monitoring of existing and future drugs. Volatile and intravenous general anaesthetic agents (GAs) are now known to exert their effects on a variety of protein targets, the most important of which seem to be the neuronal ion channels. It is hence unlikely that anaesthetic effect is the result of a unitary mechanism at the single cell level. However, by altering the behavior of ion channels GAs are believed to change the overall dynamics of distributed networks of neurons. This disruption of regular network activity can be hypothesized to cause the hypnotic and analgesic effects of GAs and may well present more stereotypical characteristics than its underlying microscopic causes. Nevertheless, there have been surprisingly few theories that have attempted to integrate, in a quantitative manner, the empirically well documented alterations in neuronal ion channel behavior with the corresponding macroscopic effects. Here we outline one such approach, and show that a range of well documented effects of anaesthetics on the electroencephalogram (EEG) may be putatively accounted for. In particular we parameterize, on the basis of detailed empirical data, the effects of halogenated volatile ethers (a clinically widely used class of general anaesthetic agent). The resulting model is able to provisionally account for a range of anaesthetically induced EEG phenomena that include EEG slowing, biphasic changes in EEG power, and the dose dependent appearance of anomalous ictal activity, as well as providing a basis for novel approaches to monitoring brain function in both health and disease.

Understanding the effects of anesthetic agents on the EEG through neural field theory

Anesthetic and analgesic agents act through a diverse range of pharmacological mechanisms. Existing empirical data clearly shows that such "microscopic" pharmacological diversity is reflected in their "macroscopic" effects on the human electroencephalogram (EEG). Based on a detailed mesoscopic neural field model we theoretically posit that anesthetic induced EEG activity is due to selective parametric changes in synaptic efficacy and dynamics. Specifically, on the basis of physiologically constrained modeling, it is speculated that the selective modification of inhibitory or excitatory synaptic activity may differentially effect the EEG spectrum. Such results emphasize the importance of neural field theories of brain electrical activity for elucidating the principles whereby pharmacological agents effect the EEG. Such insights will contribute to improved methods for monitoring depth of anesthesia using the EEG.

HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain

Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug-induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.

Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.

Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ(2), PGA(2), and PGA(1), formed by dehydration of their respective parent PGs, PGD(2), PGE(2), and PGE(1), possess a highly reactive alpha,beta-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1(+/+)) or deficient (TRPA1(-/-)) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1(+/+), but not of TRPA1(-/-) mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1(+/+) but not in TRPA1(-/-) mice. The classical proalgesic PG, PGE(2), caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1(+/+) and TRPA1(-/-) mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.