TRPs in Tox: Involvement of Transient Receptor Potential-Channels in Chemical-Induced Organ Toxicity-A Structured Review.

Chemicals can exhibit significant toxic properties. While for most compounds, unspecific cell damaging processes are assumed, a plethora of chemicals exhibit characteristic odors, suggesting a more specific interaction with the human body. During the last few years, G-protein-coupled receptors and especially chemosensory ion channels of the transient receptor potential family (TRP channels) were identified as defined targets for several chemicals. In some cases, TRP channels were suggested as being causal for toxicity. Therefore, these channels have moved into the spotlight of toxicological research. In this review, we screened available literature in PubMed that deals with the role of chemical-sensing TRP channels in specific organ systems. TRPA1, TRPM and TRPV channels were identified as essential chemosensors in the nervous system, the upper and lower airways, colon, pancreas, bladder, skin, the cardiovascular system, and the eyes. Regarding TRP channel subtypes, A1, M8, and V1 were found most frequently associated with toxicity. They are followed by V4, while other TRP channels (C1, C4, M5) are only less abundantly expressed in this context. Moreover, TRPA1, M8, V1 are co-expressed in most organs. This review summarizes organ-specific toxicological roles of TRP channels.

Zinc chloride-induced TRPA1 activation does not contribute to toxicity in vitro.

The hygroscopic zinc chloride (ZnCl2) is often used to generate smoke screens. Severe adverse pulmonary health effects have been associated with inhalation of ZnCl2 smokes. The underlying molecular toxicology is not known. Recent studies have shown that the Transient Receptor Potential Channel A1 (TRPA1) is important for sensing toxic chemicals. TRPA1 was shown to be activated by Zn2+ which was linked to pain and inflammation. In the present study, we investigated whether TRPA1 activation contributes to ZnCl2-mediated toxicity in vitro. HEK wildtype (HEK-wt), TRPA1 overexpressing HEK (HEK-A1) and A549 lung cells, endogenously expressing TRPA1, were exposed to ZnCl2. Changes of intracellular calcium levels [Ca2+]i and cell viability were assessed after ZnCl2 exposure in all cell types, without or with TRPA1 inhibition. ZnCl2 increased [Ca2+]i through TRPA1 channels in a complex manner in both HEK-A1 and A549 cells while HEK-wt did not respond to ZnCl2. There was no difference in toxicity between HEK-wt and HEK-A1 cells after ZnCl2 exposure. Inhibition of TRPA1 did not influence toxicity in all investigated cells. Thus, our in vitro results support the assumption that TRPA1 does not primarily mediate toxicity of ZnCl2 and does probably not represent a therapeutic target to abate ZnCl2 toxicity.

N-Acetyl-L-cysteine inhibits sulfur mustard-induced and TRPA1-dependent calcium influx.

Transient receptor potential family channels (TRPs) have been identified as relevant targets in many pharmacological as well as toxicological studies. TRP channels are ubiquitously expressed in different tissues and act among others as sensors for different external stimuli, such as mechanical stress or noxious impacts. Recent studies suggest that one member of this family, the transient receptor potential ankyrin 1 cation channel (TRPA1), is involved in pain, itch, and various diseases, suggesting TRPA1 as a potential therapeutic target. As a nociceptor, TRPA1 is mainly activated by noxious or electrophilic compounds, including alkylating substances. Previous studies already revealed an impact of 2-chloroethyl-ethyl sulfide on the ion channel TRPA1. In this study, we demonstrate that sulfur mustard (bis-(2-chloroethyl) sulfide, SM) activates the human TRPA1 (hTRPA1) in a dose-dependent manner measured by the increase in intracellular Ca2+ concentration ([Ca2+]i). Besides that, SM-induced toxicity was attenuated by antioxidants. However, very little is known about the underlying mechanisms. Here, we demonstrate that N-acetyl-L-cysteine (NAC) prevents SM-induced hTRPA1-activation. HEK293-A1-E cells, overexpressing hTRPA1, show a distinct increase in [Ca2+]i immediately after SM exposure, whereas this increase is reduced in cells pretreated with NAC in a dose-dependent manner. Interestingly, glutathione, although being highly related to NAC, did not show an effect on hTRPA1 channel activity. Taken together, our results provide evidence that SM-dependent activation of hTRPA1 can be diminished by NAC treatment, suggesting a direct interaction of NAC and the hTRPA1 cation channel. Our previous studies already showed a correlation of hTRPA1-activation with cell damage after exposure to alkylating agents. Therefore, NAC might be a feasible approach mitigating hTRPA1-related dysregulations after exposure to SM.

Activation of the chemosensing transient receptor potential channel A1 (TRPA1) by alkylating agents.

The transient receptor potential ankyrin 1 (TRPA1) cation channel is expressed in different tissues including skin, lung and neuronal tissue. Recent reports identified TRPA1 as a sensor for noxious substances, implicating a functional role in the molecular toxicology. TRPA1 is activated by various potentially harmful electrophilic substances. The chemical warfare agent sulfur mustard (SM) is a highly reactive alkylating agent that binds to numerous biological targets. Although SM is known for almost 200 years, detailed knowledge about the pathophysiology resulting from exposure is lacking. A specific therapy is not available. In this study, we investigated whether the alkylating agent 2-chloroethyl-ethylsulfide (CEES, a model substance for SM-promoted effects) and SM are able to activate TRPA1 channels. CEES induced a marked increase in the intracellular calcium concentration ([Ca(2+)]i) in TRPA1-expressing but not in TRPA1-negative cells. The TRP-channel blocker AP18 diminished the CEES-induced calcium influx. HEK293 cells permanently expressing TRPA1 were more sensitive toward cytotoxic effects of CEES compared with wild-type cells. At low CEES concentrations, CEES-induced cytotoxicity was prevented by AP18. Proof-of-concept experiments using SM resulted in a pronounced increase in [Ca(2+)]i in HEK293-A1-E cells. Human A549 lung epithelial cells, which express TRPA1 endogenously, reacted with a transient calcium influx in response to CEES exposure. The CEES-dependent calcium response was diminished by AP18. In summary, our results demonstrate that alkylating agents are able to activate TRPA1. Inhibition of TRPA1 counteracted cellular toxicity and could thus represent a feasible approach to mitigate SM-induced cell damage.

Influence of organophosphate poisoning on human dendritic cells.

Organophosphourus compounds (OPC, including nerve agents and pesticides) exhibit acute toxicity by inhibition of acetylcholinesterase. Lung affections are frequent complications and a risk factor for death. In addition, epidemiological studies reported immunological alterations after OPC exposure. In our experiments we investigated the effects of organophosphourus pesticides dimethoate and chlorpyrifos on dendritic cells (DC) that are essential for the initial immune response, especially in the pulmonary system. DC, differentiated from the monocyte cell line THP-1 by using various cytokines (IL-4, GM-CSF, TNF-α, Ionomycin), were exposed to organophosphourus compounds at different concentrations for a 24h time period. DC were characterized by flow cytometry and immunofluorescence using typical dendritic cell markers (e.g., CD11c, CD209 and CD83). After OPC exposure we investigated cell death, the secretion profile of inflammatory mediators, changes of DC morphology, and the effect on protein kinase signalling pathways. Our results revealed a successful differentiation of THP-1 into DC. OPC exposure caused a significant concentration-dependent influence on DC: Dendrites of the DC were shortened and damaged, DC-specific cell surface markers (i.e., CD83and CD209) decreased dramatically after chlorpyrifos exposure. Interestingly, the effects caused by dimethoate were in general less pronounced. The organophosphourus compounds affected the release of inflammatory cytokines, such as IL-1ß and IL-8. The anti-inflammatory cytokine IL-10 was significantly down regulated. Protein kinases like the Akt family or ERK, which are essential for cell survival and proliferation, were inhibited by both OPC. These findings indicate that the tested organophosphourus compounds induced significant changes in cell morphology, inhibited anti-inflammatory cytokines and influenced important protein signalling pathways which are involved in regulation of apoptosis. Thus our results highlight novel aspects -apparently independent of AChE inhibition- of OPC poisoning with regard to lung toxicity. Our findings contribute to the basic understanding of pulmonary complications caused by OPC poisoning.

Use of the Cultex® Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility.

Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is considered as a serious health hazard. For a wide range of substances basic knowledge about the toxic properties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicological in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general ("3R" principle), profound in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex® RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials. Using the Cultex® RFS, human lung epithelial cells (A549 cells) were exposed to different concentrations of airborne metal dusts (nano- and microscale particles) at the air-liquid-interface (ALI). Cell viability (WST-1 assay) as a parameter of toxicity was assessed 24h after exposure with special focus on the intra- and inter-laboratory (three independent laboratories) reproducibility. Our results show the general applicability of the Cultex® RFS with regard to the requirements of the ECVAM (European Centre for the Validation of Alternative Methods) principles on test validity underlining its robustness and stability. Intra- and inter-laboratory reproducibility can be considered as sufficient if predefined quality criteria are respected. Special attention must be paid to the pure air controls that turned out to be a critical parameter for a rational interpretation of the results. Our results are encouraging and future work is planned to improve the inter-laboratory reproducibility, to consolidate the results so far and to develop a valid prediction model.