Integrate mechanistic evidence from new approach methodologies (NAMs) into a read-across assessment to characterise trends in shared mode of action.

Read-across approaches often remain inconclusive as they do not provide sufficient evidence on a common mode of action across the category members. This read-across case study on thirteen, structurally similar, branched aliphatic carboxylic acids investigates the concept of using human-based new approach methods, such as in vitro and in silico models, to demonstrate biological similarity. Five out of the thirteen analogues have preclinical in vivo studies. Three out of them induced lipid accumulation or hypertrophy in preclinical studies with repeated exposure, which leads to the read-across hypothesis that the analogues can potentially induce hepatic steatosis. To confirm the selection of analogues, the expression patterns of the induced differentially expressed genes (DEGs) were analysed in a human liver model. With increasing dose, the expression pattern within the tested analogues got more similar, which serves as a first indication of a common mode of action and suggests differences in the potency of the analogues. Hepatic steatosis is a well-known adverse outcome, for which over 55 adverse outcome pathways have been identified. The resulting adverse outcome pathway (AOP) network, comprised a total 43 MIEs/KEs and enabled the design of an in vitro testing battery. From the AOP network, ten MIEs, early and late KEs were tested to systematically investigate a common mode of action among the grouped compounds. The targeted testing of AOP specific MIE/KEs shows that biological activity in the category decreases with side chain length. A similar trend was evident in measuring liver alterations in zebra fish embryos. However, activation of single MIEs or early KEs at in vivo relevant doses did not necessarily progress to the late KE "lipid accumulation". KEs not related to the read-across hypothesis, testing for example general mitochondrial stress responses in liver cells, showed no trend or biological similarity. Testing scope is a key issue in the design of in vitro test batteries. The Dempster-Shafer decision theory predicted those analogues with in vivo reference data correctly using one human liver model or the CALUX reporter assays. The case study shows that the read-across hypothesis is the key element to designing the testing strategy. In the case of a good mechanistic understanding, an AOP facilitates the selection of reliable human in vitro models to demonstrate a common mode of action. Testing DEGs, MIEs and early KEs served to show biological similarity, whereas the late KEs become important for confirmation, as progression from MIEs to AO is not always guaranteed.

Towards grouping concepts based on new approach methodologies in chemical hazard assessment: the read-across approach of the EU-ToxRisk project.

Read-across is one of the most frequently used alternative tools for hazard assessment, in particular for complex endpoints such as repeated dose or developmental and reproductive toxicity. Read-across extrapolates the outcome of a specific toxicological in vivo endpoint from tested (source) compounds to "similar" (target) compound(s). If appropriately applied, a read-across approach can be used instead of de novo animal testing. The read-across approach starts with structural/physicochemical similarity between target and source compounds, assuming that similar structural characteristics lead to similar human hazards. In addition, similarity also has to be shown for the toxicokinetic and toxicodynamic properties of the grouped compounds. To date, many read-across cases fail to demonstrate toxicokinetic and toxicodynamic similarities. New concepts, in vitro and in silico tools are needed to better characterise these properties, collectively called new approach methodologies (NAMs). This white paper outlines a general read-across assessment concept using NAMs to support hazard characterization of the grouped compounds by generating data on their dynamic and kinetic properties. Based on the overarching read-across hypothesis, the read-across workflow suggests targeted or untargeted NAM testing also outlining how mechanistic knowledge such as adverse outcome pathways (AOPs) can be utilized. Toxicokinetic models (biokinetic and PBPK), enriched by in vitro parameters such as plasma protein binding and hepatocellular clearance, are proposed to show (dis)similarity of target and source compound toxicokinetics. Furthermore, in vitro to in vivo extrapolation is proposed to predict a human equivalent dose, as potential point of departure for risk assessment. Finally, the generated NAM data are anchored to the existing in vivo data of source compounds to predict the hazard of the target compound in a qualitative and/or quantitative manner. To build this EU-ToxRisk read-across concept, case studies have been conducted and discussed with the regulatory community. These case studies are briefly outlined.

Investigating cell type specific mechanisms contributing to acute oral toxicity.

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 98 were identified with target organ specific effects, of which 93% were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% as non-toxic. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

Adverse outcome pathways: opportunities, limitations and open questions.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

The virtual cell based assay: Current status and future perspectives.

In order to replace the use of animals in toxicity testing, there is a need to predict in vivo toxic doses from concentrations that cause toxicological effects in relevant in vitro systems. The Virtual Cell Based Assay (VCBA) estimates time-dependent concentration of a test chemical in the cell and cell culture for a given in vitro system. The concentrations in the different compartments of the cell and test system are derived from ordinary differential equations, physicochemical parameters of the test chemical and properties of the cell line. The VCBA has been developed for a range of cell lines including BALB/c 3T3 cells, HepG2, HepaRG, lung A459 cells, and cardiomyocytes. The model can be used to design and refine in vitro experiments and extrapolate in vitro effective concentrations to in vivo doses that can be applied in risk assessment. In this paper, we first discuss potential applications of the VCBA: i) design of in vitro High Throughput Screening (HTS) experiments; ii) hazard identification (based on acute systemic toxicity); and iii) risk assessment. Further extension of the VCBA is discussed in the second part, exploring potential application to i) manufactured nanomaterials, ii) additional cell lines and endpoints, and considering iii) other opportunities.

Alternative approaches for identifying acute systemic toxicity: Moving from research to regulatory testing.

Acute systemic toxicity testing provides the basis for hazard labeling and risk management of chemicals. A number of international efforts have been directed at identifying non-animal alternatives for in vivo acute systemic toxicity tests. A September 2015 workshop, Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing, reviewed the state-of-the-science of non-animal alternatives for this testing and explored ways to facilitate implementation of alternatives. Workshop attendees included representatives from international regulatory agencies, academia, nongovernmental organizations, and industry. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements. Attendees particularly noted the need to characterize variability in reference data to evaluate new approaches. They also noted the importance of understanding the mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Workshop breakout groups explored different approaches to reducing or replacing animal use for acute toxicity testing, with each group crafting a roadmap and strategy to accomplish near-term progress. The workshop steering committee has organized efforts to implement the recommendations of the workshop participants.

Exploring waiving opportunities for mammalian acute systemic toxicity tests.

A survey was carried out to explore opportunities for waiving mammalian acute systemic toxicity tests. We were interested in finding out whether data from a sub-acute toxicity test could be used to predict the outcome of an acute systemic toxicity test. The survey was directed at experts in the field of toxicity testing, and was carried out in the context of the upcoming 2018 final registration deadline for chemicals under the EU REACH Regulation. In addition to the survey, a retrospective data analysis of chemicals that had already been registered with the European Chemicals Agency, and for which both acute and sub-acute toxicity data were available, was carried out. This data analysis was focused on chemicals that were administered via the oral route. The answers to the questionnaire showed a willingness to adopt waiving opportunities. In addition, the responses showed that data from a sub-acute toxicity test or dose-range finding study might be useful for predicting chemicals that do not require classification for acute oral toxicity (LD50 > 2000mg/kg body weight). However, with the exception of substances that fall into the non-classified category, it is difficult to predict current acute oral toxicity categories.

Involvement of Mast Cells in α7 Nicotinic Receptor Agonist Exacerbation of Freund's Complete Adjuvant-Induced Monoarthritis in Mice.

OBJECTIVE: Activation of antiinflammatory cholinergic (vagal) pathways can reduce inflammation, and in vitro studies support a pivotal role of α7 nicotinic acetylcholine receptors (α7-nAChR), macrophages, and T cells in these events. The aim of this study was to assess α7-nAChR agonists as an antiinflammatory treatment for Freund's complete adjuvant (CFA)-induced monoarthritis. METHODS: Arthritis was induced by intraarticular injection of CFA unilaterally into the knee joints of mice. Animals were treated with α7-nAChR agonists (AR-R17779 or A844606), with or without antagonists (COG133 or methyllycaconitine), and joint inflammation and pain were assessed. Experiments were repeated in c-Kit(W-sh) mast cell-deficient mice, and the effects of an α7-nAChR agonist on mast cell proliferation, migration, and activation by lipopolysaccharide (LPS) were tested. RESULTS: Treatment with α7-nAChR agonists significantly exacerbated CFA-induced arthritis and pain, as gauged by all indices of assessment, the specificity of which was confirmed by coadministration of an nAChR antagonist that attenuated the increase in disease severity. Toluidine blue-positive mast cells were increased in the joint capsule of CFA plus AR-R17779-treated mice, and AR-R17779 enhanced LPS-induced TNF proliferation and migration of a human mast cell line. The AR-R17779-driven increase in severity of CFA-induced arthritis was significantly reduced in mast cell-deficient mice. CONCLUSION: Using CFA to elicit a local inflammatory response, we found that pharmacologic activation of α7-nAChR exacerbated joint inflammation and pain, in part via mast cells, which illustrates the organ- and disease-specific nature of regulatory neuroimmune mechanisms. Thus, α7-nAChR activation may not be uniformly antiinflammatory in all types of inflammatory joint disease.

Splenic B cells from Hymenolepis diminuta-infected mice ameliorate colitis independent of T cells and via cooperation with macrophages.

Helminth parasites provoke multicellular immune responses in their hosts that can suppress concomitant disease. The gut lumen-dwelling tapeworm Hymenolepis diminuta, unlike other parasites assessed as helminth therapy, causes no host tissue damage while potently suppressing murine colitis. With the goal of harnessing the immunomodulatory capacity of infection with H. diminuta, we assessed the putative generation of anti-colitic regulatory B cells following H. diminuta infection. Splenic CD19(+) B cells isolated from mice infected 7 [HdBc(7(d))] and 14 d (but not 3 d) previously with H. diminuta and transferred to naive mice significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-, oxazolone-, and dextran-sodium sulfate-induced colitis. Mechanistic studies with the DNBS model, revealed the anti-colitic HdBc(7(d)) was within the follicular B cell population and its phenotype was not dependent on IL-4 or IL-10. The HdBc(7(d)) were not characterized by increased expression of CD1d, CD5, CD23, or IL-10 production, but did spontaneously, and upon LPS plus anti-CD40 stimulation, produce more TGF-ß than CD19(+) B cells from controls. DNBS-induced colitis in RAG1(-/-) mice was inhibited by administration of HdBc(7(d)), indicating a lack of a requirement for T and B cells in the recipient; however, depletion of macrophages in recipient mice abrogated the anti-colitic effect of HdBc(7(d)). Thus, in response to H. diminuta, a putatively unique splenic CD19(+) B cell with a functional immunoregulatory program is generated that promotes the suppression of colitis dominated by TH1, TH2, or TH1-plus-TH2 events, and may do so via the synthesis of TGF-ß and the generation of, or cooperation with, a regulatory macrophage.

TRPA1 is essential for the vascular response to environmental cold exposure.

The cold-induced vascular response, consisting of vasoconstriction followed by vasodilatation, is critical for protecting the cutaneous tissues against cold injury. Whilst this physiological reflex response is historic knowledge, the mechanisms involved are unclear. Here by using a murine model of local environmental cold exposure, we show that TRPA1 acts as a primary vascular cold sensor, as determined through TRPA1 pharmacological antagonism or gene deletion. The initial cold-induced vasoconstriction is mediated via TRPA1-dependent superoxide production that stimulates α2C-adrenoceptors and Rho-kinase-mediated MLC phosphorylation, downstream of TRPA1 activation. The subsequent restorative blood flow component is also dependent on TRPA1 activation being mediated by sensory nerve-derived dilator neuropeptides CGRP and substance P, and also nNOS-derived NO. The results allow a new understanding of the importance of TRPA1 in cold exposure and provide impetus for further research into developing therapeutic agents aimed at the local protection of the skin in disease and adverse climates.

Murine autoimmune arthritis is exaggerated by infection with the rat tapeworm, Hymenolepis diminuta.

Infection with helminth parasites triggers strong and stereotypic immune responses in humans and mice, which can protect against specific experimentally-induced autoimmune diseases. We have shown that infection with the rat tapeworm, Hymenolepis diminuta, confers a protective effect on FCA-induced joint inflammation. Here, we investigated the effect of a prophylactic infection with H. diminuta on the K/BxN-serum model of polyarthritis in BALB/c mice. Mice were infected with 10 cysticercoids of H. diminuta by oral gavage and 8 days later arthritis was induced by i.p. injection of K/BxN arthritogenic serum. Joint swelling and pain measurements were recorded throughout a 13 day time course. At necropsy, joints and blood serum were collected. K/BxN-treated mice developed joint inflammation in the front paws, hind paws and knees as shown by increased swelling, mechanical allodynia and myeloperoxidase activity. Mice infected with H. diminuta had more severe disease, with increased eosinophil peroxidase activity in their paws and greater inflammatory infiltrate and synovitis in the knee joints. Hymenolepis diminuta-infected mice displayed significant increases in serum levels of C5a and mast cell protease-1 compared with K/BxN-serum only treatment, the latter being indicative of mast cell activation. In contrast to the protective effect of infection with H. diminuta in FCA-induced monoarthritis, infection with this helminth exacerbated K/BxN serum-induced polyarthritis in BALB/c mice. This correlated with increases in C5a and mast cell activation: factors critical in the development of K/BxN-induced arthritis. Thus, while data accumulate from animal models showing that infection with helminth parasites may be beneficial for a variety of auto-inflammatory diseases, our findings demonstrate the potential for helminths to exacerbate disease. Hence care is needed when helminth therapy is translated into a clinical setting.

Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C.

The enteric epithelium must absorb nutrients and water and act as a barrier to the entry of luminal material into the body; this barrier function is a key component of innate immunity. Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy occurs via inhibition of prostaglandin synthesis and perturbed epithelial mitochondrial activity. Here, the direct effect of NSAIDs [indomethacin, piroxicam (cyclooxygenase 1 and 2 inhibitors), and SC-560 (a cyclooxygenase 1 inhibitor)] on the barrier function of human T84 epithelial cell line monolayers was assessed by transepithelial electrical resistance (TER) and internalization and translocation of a commensal Escherichia coli. Exposure to E. coli in the presence and absence of drugs for 16 h reduced TER; however, monolayers cotreated with E. coli and indomethacin, but not piroxicam or SC-560, displayed significant increases in internalization and translocation of the bacteria. This was accompanied by increased reactive oxygen species (ROS) production, which was also increased in epithelia treated with E. coli only. Colocalization revealed upregulation of superoxide synthesis by mitochondria in epithelia treated with E. coli + indomethacin. Addition of antioxidants (vitamin C or a green tea polyphenol, epigallocathechin gallate) quenched the ROS and prevented the increase in E. coli internalization and translocation evoked by indomethacin, but not the drop in TER. Evidence of increased apoptosis was not observed in this model. The data implicate epithelial-derived ROS in indomethacin-induced barrier dysfunction and show that a portion of the bacteria likely cross the epithelium via a transcellular pathway. We speculate that addition of antioxidants as dietary supplements to NSAID treatment regimens would reduce the magnitude of decreased barrier function, specifically the transepithelial passage of bacteria.

A distinct role for transient receptor potential ankyrin 1, in addition to transient receptor potential vanilloid 1, in tumor necrosis factor α-induced inflammatory hyperalgesia and Freund's complete adjuvant-induced monarthritis.

OBJECTIVE: To investigate the involvement of transient receptor potential ankyrin 1 (TRPA1) in inflammatory hyperalgesia mediated by tumor necrosis factor α(TNFα) and joint inflammation. METHODS: Mechanical hyperalgesia was assessed in CD1 mice, mice lacking functional TRP vanilloid 1 (TRPV1-/-) or TRPA1 (TRPA1-/-), or respective wildtype (WT) mice. An automated von Frey system was used, following unilateral intraplantar injection of TNFα or intraarticular injection of Freund's complete adjuvant (CFA). Knee swelling and histologic changes were determined in mice treated with intraarticular injections of CFA. RESULTS: TNFα induced cyclooxygenase-independent bilateral mechanical hyperalgesia in CD1 mice. The selective TRPV1 receptor antagonist SB-366791 had no effect on mechanical hyperalgesia when it was coinjected with TNFα, but intrathecally administered SB- 366791 attenuated bilateral hyperalgesia, indicating the central but not peripheral involvement of TRPV1 receptors. A decrease in pain sensitivity was also observed in TRPV1-/- mice. Intraplantar coadministration of the TRPA1 receptor antagonist AP-18 with TNFα inhibited bilateral hyperalgesia. Intrathecal treatment with AP-18 also reduced TNFα-induced hyperalgesia. CFA-induced mechanical hyperalgesia in CD1 mice was attenuated by AP-18 (administered by intraarticular injection 22 hours after the administration of CFA). Furthermore, intraarticular CFA­induced ipsilateral mechanical hyperalgesia was maintained for 3 weeks in TRPA1 WT mice. In contrast, TRPA1-/- mice exhibited mechanical hyperalgesia for only 24 hours after receiving CFA. CONCLUSION: Evidence suggests that endogenous activation of peripheral TRPA1 receptors plays a critical role in the development of TNFα-induced mechanical hyperalgesia and in sustaining the mechanical hyperalgesia observed after intraaarticular injection of CFA. These results suggest that blockade of TRPA1 receptors may be beneficial in reducing the chronic pain associated with arthritis.

4-oxo-2-nonenal (4-ONE): evidence of transient receptor potential ankyrin 1-dependent and -independent nociceptive and vasoactive responses in vivo.

This study explores the in vivo effects of the proposed transient receptor potential ankyrin 1 (TRPA1) agonist 4-oxo-2-nonenal (4-ONE). Pharmacological inhibitors and genetically modified mice were used to investigate the ability of 4-ONE to act via TRPA1 receptors and possible mechanisms involving transient receptor potential vanilloid 1 (TRPV1). We hypothesized that 4-ONE activates sensory nerves, via TRPA1 or possibly TRPV1, and thus triggers mechanical hyperalgesia, edema formation, and vasodilatation in mice. An automated dynamic plantar aesthesiometer was used to determine hind paw withdrawal thresholds, and a laser Doppler flowmeter was used to measure skin blood flow. Edema formation was determined by measuring paw weights and thickness. 4-ONE (10 nmol) triggers unilateral mechanical hyperalgesia, edema formation, and vasodilatation in mice and is shown here to exhibit TRPA1-dependent and -independent effects. Neurogenic vasodilatation and mechanical hyperalgesia at 0.5 h postinjection were significantly greater in TRPA1 wild-type (WT) mice compared with TRPA1 knockout (KO) mice. Edema formation throughout the time course as well as mechanical hyperalgesia from 1 to 4 h postinjection were similar in WT and TRPA1 KO mice. Studies involving TRPV1 KO mice revealed no evidence of TRPV1 involvement or interactions between TRPA1 and TRPV1 in mediating the in vivo effects of 4-ONE. Previously, 4-ONE was shown to be a potent TRPA1 agonist in vitro. We demonstrate its ability to mediate vasodilatation and certain nociceptive effects in vivo. These data indicate the potential of TRPA1 as an oxidant sensor for vasodilator responses in vivo. However, 4-ONE also triggers TRPA1-independent effects that relate to edema formation and pain.

Evidence for the pathophysiological relevance of TRPA1 receptors in the cardiovascular system in vivo.

AIMS: The aim of the study is to investigate transient receptor potential ankyrin 1 (TRPA1)-induced responses in the vasculature and on blood pressure and heart rate (HR), in response to TRPA1 agonists using wild-type (WT) and TRPA1 knockout (KO) mice. METHODS AND RESULTS: TRPA1 agonists allyl isothiocyanate and cinnamaldehyde (CA) significantly increased blood flow in the skin of anaesthetized WT, but not in TRPA1 KO mice. CA also induced TRPA1-dependent relaxation of mesenteric arteries. Intravenously injected CA induced a transient hypotensive response accompanied by decreased HR that was, depending on genotype and dose, followed by a more sustained dose-dependent pressor response (10-320 micromol/kg). CA (80 micromol/kg) induced a depressor response that was significantly less in TRPA1 KO mice, with minimal pressor effects. The pressor response of a higher CA dose (320 micromol/kg) was observed in WT but not in TRPA1 KO mice, indicating involvement of TRPA1. Experiments using TRP vanilloid 1 (TRPV1) KO and calcitonin gene-related peptide (CGRP) KO mice provided little evidence for the involvement of TRPV1 or CGRP, nor did blocking substance P receptors affect responses. However, the cholinergic antagonist atropine sulphate (5 mg/kg) significantly inhibited the depressor response and slowed HR with CA (80 micromol/kg), but had no effect on pressor responses. The pressor response remained unaffected, even in the presence of the ganglion blocker hexamethonium bromide (1 mg/kg). The alpha-adrenergic blocker prazosin hydrochloride (1 mg/kg) significantly inhibited both components, but not slowed HR. CONCLUSION: TRPA1 is involved in mediating vasodilation. TRPA1 can also influence changes in blood pressure of possible relevance to autonomic system reflexes and potentially to vasovagal/neurocardiogenic syncope disorders.

Hydrogen peroxide is a novel mediator of inflammatory hyperalgesia, acting via transient receptor potential vanilloid 1-dependent and independent mechanisms.

Inflammatory diseases associated with pain are often difficult to treat in the clinic due to insufficient understanding of the nociceptive pathways involved. Recently, there has been considerable interest in the role of reactive oxygen species (ROS) in inflammatory disease, but little is known of the role of hydrogen peroxide (H(2)O(2)) in hyperalgesia. In the present study, intraplantar injection of H(2)O(2)-induced a significant dose- and time-dependent mechanical and thermal hyperalgesia in the mouse hind paw, with increased c-fos activity observed in the dorsal horn of the spinal cord. H(2)O(2) also induced significant nociceptive behavior such as increased paw licking and decreased body liftings. H(2)O(2) levels were significantly raised in the carrageenan-induced hind paw inflammation model, showing that this ROS is produced endogenously in a model of inflammation. Moreover, superoxide dismutase and catalase significantly reduced carrageenan-induced mechanical and thermal hyperalgesia, providing evidence of a functionally significant endogenous role. Thermal, but not mechanical, hyperalgesia in response to H(2)O(2) (i.pl.) was longer lasting in TRPV1 wild type mice compared to TRPV1 knockouts. It is unlikely that downstream lipid peroxidation was increased by H(2)O(2). In conclusion, we demonstrate a notable effect of H(2)O(2) in mediating inflammatory hyperalgesia, thus highlighting H(2)O(2) removal as a novel therapeutic target for anti-hyperalgesic drugs in the clinic.

A reactive oxygen species-mediated component in neurogenic vasodilatation.

AIMS: Activation of the transient receptor potential vanilloid receptor 1 (TRPV1) leads to release of potent microvascular vasodilator neuropeptides. This study was designed to investigate in vivo mechanisms involved in TRPV1-mediated peripheral vasodilatation. METHODS AND RESULTS: Wildtype (WT) and TRPV1 knockout (KO) mice were investigated in a model of peripheral vasodilatation. Blood flow was measured by laser Doppler flowmetry under anaesthesia and following local application of the TRPV1 agonist capsaicin. A sustained (60 min) increase in blood flow was observed in WT but not TRPV1 KO mouse ears. This response was resistant to blockers of classic vasodilators but inhibited in pharmacogenetic experiments that targeted blockade of the substance P (SP) and calcitonin gene-related peptide (CGRP) pathways. The TRPV1-mediated vasodilatation was also attenuated by treatment with superoxide dismutase and the hydrogen peroxide scavenger catalase, but not by deactivated enzymes, supporting a novel role for reactive oxygen species (ROS) generation. Furthermore, neurogenic vasodilatation was observed neither in the presence of the selective NADPH inhibitor apocynin, nor in gp91 phox KO mice, under conditions where prostaglandin E1-induced vasodilatation occurred. Finally, a role of neuropeptides in initiating a ROS-dependent component was verified as superoxide dismutase, catalase, and apocynin inhibited SP and CGRP vasodilatation. CONCLUSION: These studies provide in vivo evidence that ROS are involved in mediating TRPV1- and neuropeptide-dependent neurogenic vasodilatation. An essential role of NADPH oxidase-dependent ROS is revealed that may be of fundamental importance to the neurogenic vasodilator component involved in circulatory homeostasis and the pathophysiology of certain cardiovascular diseases.