ToxSci Advance Access originally published online on June 16, 2007
Toxicological Sciences 2007 99(1):174-180; doi:10.1093/toxsci/kfm164
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Surfactant-Induced TRPV1 Activity—A Novel Mechanism for Eye Irritation?
Department of Neurochemistry, Stockholm University, SE-106 91 Stockholm, Sweden
1 To whom correspondence should be addressed at Department of Neurochemistry, Stockholm University, Svante Arrhenius v. 21 A, SE-106 91 Stockholm, Sweden. Fax: +46-(8)1-61-371. E-mail: johanna{at}neurochem.su.se.
Received March 13, 2007; accepted June 11, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The pain receptor transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca2+ influx with secondary effects leading to neurogenic inflammation. Here we report specific activation of TRPV1 by detergent-containing hygiene products measured as intracellular Ca2+ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. Children products marketed as "painless" (containing lower concentration of detergents), and conditioners (without detergents) did not induce specific TRPV1 activation. Furthermore, low concentrations of the detergent sodium lauryl sulfate dose-dependently induced Ca2+ influxes that could be addressed to TRPV1. These results reveal a novel mechanistic pathway for surfactant-induced nociception, which may be an important endpoint in in vitro test batteries as alternatives to Draize's rabbit eye test for classification of eye irritating products.
Key Words: pain; detergent; TRPV1; eye; in vitro; SH-SY5Y.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The sensory neurons are the outermost sensors informing us of the surrounding environment by the activation of surface receptors receptive to mechanical force, temperature, and chemical stimuli. Free nerve endings are thought to convey painful signals transmitted by unmyelinated C-fibers or thinly myelinated A
-fibers (Julius and Basbaum, 2001
; Sternini, 1997). One compound known since ancient times to be highly irritating is capsaicin, the pungent component in chili peppers (reviewed in Holzer, 1991). Contact of capsaicin with eyes or ruptured skin causes a painful burning sensation accompanied by an inflammatory response in surrounding tissues (Geppetti and Holzer, 1996). The capsaicin receptor TRPV1 belongs to the temperature-sensitive ion-gated channels inducing Ca2+ (and Na+) influxes when activated (Caterina et al., 1997). TRPV1 (transient receptor potential vanilloid type 1) is predominantly located in the C-fiber associated dorsal root ganglion and trigeminal ganglion neurons and is activated by pungent chemicals, noxious temperatures, and low pH (Caterina et al., 1997; Tominaga et al., 1998). Furthermore, the receptor is a key mediator of neurogenic inflammation (Planells-Cases et al., 2005). The sensation of detergent containing hygiene products, lemon, or chili peppers entering the eyes or a wound is the same, i.e., a burning stinging pain, suggesting a mutual mechanism.
In order to protect consumers, daily used products must be classified regarding their corrosive or irritating properties before entering the market. Today, the heavily criticized Draize's rabbit eye test is used (Draize et al., 1944). In this test neurogenic inflammation is one contributor to the visible effects in the conjunctiva and corneal tissues. The development of several in vitro methods for assessment of eye irritation and corrosion is moving forward in an attempt to replace the Draize in vivo test (Abbott, 2005; Balls et al., 1999; Curren and Harbell, 1998; Van Goethem et al., 2006). However, no in vitro alternatives include a neuronal endpoint. The cornea, eyelid, and the conjunctiva are extensively innervated by A- and C-fibers (Marfurt, 2000), neurons known to express TRPV1. These facts together with the ability of multiple chemicals to activate TRPV1, suggest that a cheap, user-friendly, neuronal model predicting chemically induced nociception with a clear mechanistic approach could be highly valuable for classification of eye irritants (Garle and Fry, 2003; Lilja and Forsby, 2004).
Herein, we show that hygiene products with a high content of detergents specifically activated TRPV1, whereas conditioners and children products marketed as "painless" (containing no or a low concentration of detergents) did not. Furthermore, we show that the widely used detergent sodium lauryl sulfate (SLS) dose-dependently induced TRPV1-specific Ca2+ influxes in TRPV1-SH-SY5Y cells but no Ca2+ influx was detected in native SH-SY5Y cells.
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MATERIALS AND METHODS |
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All chemicals in this study were obtained from Sigma-Aldrich (St Louis, MO and CA) unless stated otherwise.
Cells.
The human neuroblastoma cell line SH-SY5Y (a kind gift from Eewa Nånberg, Department of Genetics and Pathology, Uppsala University, Sweden) was stably transfected with a TRPV1-expressing plasmid with the aim to generate a cheap, reproducible neuronal-like sensory cell model. The pCAGIPuro plasmid (a gift from Dr Johan Lundkvist at Karolinska Institute, Stockholm, Sweden) was linearized with BstXI (5') and NotI (3') for insertion of the trpv1-gene amplified by PCR from the trpv1(rat)-pcDNA3 plasmid (kindly provided by Dr David Julius at the University of California, San Francisco, CA) with the sense primer: ATC GAT TCC ATA GCG TTG GAT GGA ACA ACG GGC TAG CTT; and antisense primer: ATC GAT TGC GGC CGC TTA TTT CTC CCC TGG GAC CA. The trpv1-pCAGIPuro plasmid was introduced to cells using lipofectamine 2000 according to the manufacturer's instructions and the cells were selected for puromycin resistance (all chemicals for cell culturing and cloning were obtained from Invitrogen, Carlsbad, CA). Cells are stored under liquid nitrogen and used for 10 generations from defreezing, routinely screened for mycoplasma infection.
Both the native SH-SY5Y and the TRPV1-SH-SY5Y cells were maintained as monolayer in 75-cm2 flasks (Corning Incorporated, Corning, NY) and routinely subcultivated by trypsination (0.05% trypsin and 0.02% ethylenediaminetetraacetic acid [EDTA] in Hank's salt solution) once a week and plated in cell medium (Earle's minimum essential medium [EMEM] with Earle's salts supplemented with 10% fetal calf serum, 1% nonessential amino acids, 2mM L-glutamine, 100 µg streptomycin/ml, 100 units penicillin/ml, and for the TRPV1-expressing cells 0.4 µg/ml puromycin was added, all from Invitrogen). Medium was changed every 3–4 days and the cells were kept at 37°C in a humidified atmosphere with 5% CO2.
Immunohistochemistry.
The puromycin-selected TRPV1-SH-SY5Y clones were analyzed for TRPV1 expression. TRPV1 and native SH-SY5Y cells were cultured on cover slips and fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) followed by permeabilization in 100% methanol. Unspecific binding was blocked with 2% bovine serum albumin (BSA) in PBS. Following 7 min nuclear staining with Hoechst 33258 (diluted 1:10,000 in 2% BSA/PBS), the cells were hybridized with primary rabbit anti-TRPV1 antibodies (diluted 1:1,000 in 2% BSA/PBS) (C-terminal residues 824–838, Alomone laboratories, Jerusalem, Israel) for 30 min, quickly rinsed in 2% BSA/PBS, and blotted with secondary Alexa fluor red 568-conjugated antibodies (goat anti-rabbit IgG antibody Molecular Probes, Eugene, OR; diluted 1:1000 in 2% BSA/PBS) for 30 min followed by a quick rinse in 2% BSA/PBS. Fluorescence microscopy was performed using Leica DMIRE2/DC350F image work station.
Western blot.
Cells were rinsed in PBS supplemented with 0.9mM CaCl2 and 0.5mM MgCl2 and proteins extracted in 75-µl hypotonic lysis buffer (50mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 20mM EDTA, 1% Triton X-100, 10mM NaF, 30mM Na4PPi, 0.3 mg/ml Benzamidine and 1mM NaVO4) for 30 min. The proteins were clarified by short centrifugation at 20,000 x g and the concentration in the supernatant was determined using the Bio Rad Protein Assay (Bio Rad Laboratories, Inc., Hercules, CA). Proteins (15 µg) in sample buffer (62.5mM Tris–HCl [pH 6.8], 2% SLS, 25mM dithiotreitol, 10% glycerol, and bromophenol blue) were separated on a 7% sodium dodecyl sulfate–polyacrylamide gel. The proteins, transferred to a nitrocellulose membrane, were stained with Ponceau S solution to verify even loading. Unspecific immunoblotting was blocked with 4% milk powder in Tween-20 (0.2%) containing PBS (referred to as blocking solution) and TRPV1 proteins were blotted with rabbit anti-TRPV1 antibodies (Alomone laboratories, Jerusalem, Israel) 1:10,000 in blocking solution at 4°C for 16 h. Following 4 x 15 min washings in blocking solution the membrane was incubated with horse radish peroxidase–conjugated anti-rabbit IgG antibodies (Amersham Biosciences, Buckinghamshire, UK) 1:3000 in blocking solution for 1 h at room temperature. After several washings in 0.2% Tween-20–containing PBS, a specific 95-kDa band (corresponding to TRPV1) as well as 150- and 75- to 80-kDa bands were visualized with luminescence (Perkin Elmer Life sciences, Boston, MA) on hyperfilms (Amersham Biosciences).
Intracellular Ca2+ measurements.
Prior to Ca2+ measurements the cells were seeded at a cell density of 20,000 cells/well 3–4 days from measurements to yield 90% confluence in 96-well plates. Acute cellular increase in Ca2+ levels was measured in a Flex Station II semi-HTS fluorescence reader (Molecular Devices, Corp., Sunnyvale, CA) using the cell permeable fluorescent probe Fura-2/AM. The cells were incubated with 2µM Fura-2/AM for 30 min in 37°C followed by two washings in HEPES–Krebs–Ringer (HKR) buffer (125mM NaCl, 5mM KCl, 1.2mM MgSO4 x 7H2O , 2mM CaCl2, 1.2mM KH2PO4 x 2H2O, 25mM HEPES [free acid], and 6mM glucose, pH 7.4) followed by incubation in HKR buffer for 30 min. The ratio of 340(Ca2+-bound Fura-2)/380(Fura-2) nm excitatory wavelengths was measured without interruption before (basal level) and during the 2-min exposure to the test products or capsaicin. The ratio values after addition of the chemicals reached the peak value instantly and did not change within the 2 min, hence a mean value could be calculated. The mean value (% increase of basal free intracellular Ca2+ level) from triplicates in a 96-well plate was presented for each concentration from each experiment. The TRPV1 antagonist capsazepine (Bevan et al., 1992) (10µM) was added simultaneously with each concentration of the chemicals in triplicates as negative control. For the TRPV1-SH-SY5Y cells the intracellular Ca2+ increase induced by 10nM of the specific TRPV1-agonist capsaicin was set to maximal response at each experiment and the effect of the test products was calculated as % of maximal response (10nM capsaicin is exclusively TRPV1-specific and can be blocked by capsazepine in this cell line as viewed in Fig. 3). As control for unspecific membrane leakage, the native non-TRPV1–expressing SH-SY5Y cells were tested for SLS-induced Ca2+ influx identically to the TRPV1-SH-SY5Y cells except that the % Ca2+ increase was naturally not adjusted for capsaicin (maximal TRPV1 response). All test products used were diluted to homogenous solutions in HKR buffer (wt/vol) as indicated in the figures and the addition to the cells was performed robotically during measurements by the Flex Station II reader.
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Products.
The test products used in this study were: Herbal essence shampoo "Rainforest flowers" (Midelfart/Procter & Gamble United Kingdom, Weybridge, Surrey, UK); Natusan kids shampoo "Alien apple" (Johnson & Johnson, Düsseldorf, Germany); Respons natural shampoo "oliveoil & lemon" (Garnier, Paris, France); Dove essential care shampoo (Lever Fabergé, Dublin, Ireland); Natusan kids conditioner spray "Star shine" (Johnson & Johnson, Düsseldorf, Germany); Respons natural conditioner "oliveoil & lemon" (Garnier, Paris, France); Palmolive aroma therapy shower gel, with grapefruit, orange and cedarwood, (Colgate-Palmolive, Mumbai, India); Lactacyd soap (GlaxoSmithKline, Ballerup, Denmark); Farena liquid soap (Sterisol, Vadstena, Sweden); Frøya liquid soap (Nilfisk-Advance A/S, Brøndby, Denmark); SLS (Scharlau Chemie S.A Barcelona, Spain).
Statistical analysis.
Graphs were constructed in GraphPad Prism 4 software and data were analyzed by Student's unpaired t-test (two-tailed), the effect of each concentration compared to the effect of the same concentration coincubated with capsazepine.
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RESULTS |
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Analysis of Clones
Approximately five cell clones survived the effective selection procedure in Puro-EMEM and these clones were mixed to one population and analyzed for expression and functionality. All cloned cells analyzed, possessed equal TRPV1 expression as visualized with immunofluorescence while the native SH-SY5Y cells did not express TRPV1 (Figs. 1e–f). Western blot technique also viewed the high expression of TRPV1 receptors in the TRPV1-SH-SY5Y cells absent in the native cells (Fig. 2). Morphologically, the cloned cells did not change as compared to their native counterparts (Figs. 1a and 1b). To analyze the functionality of the TRPV1 receptors, a concentration–effect curve of capsaicin ranging from 0.1 to 10,000nM was determined by measuring acute intracellular Ca2+ increase using the fluorescing and Ca2+-binding probe Fura-2/AM. To block the capsaicin response, 10µM of the TRPV1 antagonist capsazepine was used (Fig. 3). The maximal response of about 200% increase in intracellular free Ca2+ (from basal level), that could still be blocked by capsazepine, was reached at 50nM capsaicin.
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TRPV1 Activity
The aim of establishing this stably TRPV1-expressing neuroblastoma cell line was to investigate a possible TRPV1 mechanism for surfactant-induced mild eye nociception as proposed before (Lilja and Forsby, 2004
). The commercial hygiene products were applied to the cells in diluted solutions and stimulation was monitored during exposure as an increase in the intracellular free Ca2+ levels using a semi-HTS fluorometer for 96-well plates. All the regular shampoos and soaps induced a dose-dependent TRPV1 mediated Ca2+ influx that induced 50–100% of maximal Ca2+ response at 0.005% and 0.01% (wt/vol) that was significantly (although not completely) blocked by the TRPV1 antagonist capsazepine (Figs. 4a–h). Only the soaps Farena and Lactacyd could induce a TRPV1-specific Ca2+ increase also at 0.05% (wt/vol) which was significantly blocked by capsazepine and the soap Farena could induce a TRPV1-specific Ca2+ increase also at 0.1% (Figs. 4h and 4f). All other soaps and shampoos tested induced unspecific Ca2+ influxes at this and higher concentrations that could not be inhibited by capsazepine. Unspecific cytotoxicity after exposure of the higher concentrations of the products was also indicated by morphological evaluation of the cells under the microscope, showing rounded cells detaching from the cell culture surface. The shampoo for children marketed as "painless" and "no more tears" could only induce a Ca2+ influx at higher concentrations (
0.05% wt/vol) that was not addressed to TRPV1 because it could not be inhibited by capsazepine (Fig. 4d). The conditioners (both regular and for children) did not induce any Ca2+ influx at any concentration tested (Figs. 4i and 4j). To further assess the specific activation of TRPV1 by these products we sought the common ingredient of the products having this effect. All the active products contained high concentrations (first chemical placed after aqua in the ingredients list on the package) of the detergents sodium laureth sulfate, SLS or sodium C12–13 pareth sulfate. Hence, to test our hypothesis that the detergents were the main contributors to the activation of TRPV1 we exposed the TRPV1-SH-SY5Y cells to SLS. Indeed, 5–25µM SLS induced a dose-dependent specific Ca2+ influx that could be significantly blocked by capsazepine (Fig. 5a). Administering SLS to native SH-SY5Y cells did not induce any Ca2+ influx at 1–25µM (Fig. 5b) but at higher concentrations an unspecific leakage was observed, as was the case in the TRPV1-expressing cells in the presence of capsazepine (not shown). The basal Ca2+ ratio of 340/380 nm were the same for the native and the TRPV1-expressing SH-SY5Y cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Almost everyone has sometime exposed their eyes to shampoo, or chopped chili peppers and accidentally poked a finger into an eye. The smarting sensation of pain is the same, and we here show that detergent containing hygiene products induce a dose-dependent TRPV1-specific Ca2+ influx comparative to capsaicin, in the first reported neuroblastoma SH-SY5Y cell line stably expressing functional TRPV1 receptors (Lilja et al., 2007
). All products used in this study that contained high contents (as determined by placement in the ingredients list on the package) of detergents like sodium laureth sulfate, SLS or sodium C12–13 pareth sulfate, could at low concentrations induce a Ca2+ influx that was significantly (p < 0.01) inhibited by capsazepine. Moreover, we tested the frequently used detergent SLS for its TRPV1-specific effects in this cell model. Certainly, SLS could, at low concentrations and dose-dependently, activate Ca2+ influxes that was significantly (p < 0.01) blocked by capsazepine. Additionally, native non-TRPV1–expressing SH-SY5Y cells did not respond to SLS at these low concentrations. Naturally, at higher concentrations of all detergent-containing products tested the Ca2+ influx could no longer be inhibited by capsazepine, indicative of unspecific membrane leakage. The soap Farena, though, did induce a TRPV1-specific Ca2+ increase that was significantly blocked by capsazepine (p < 0.001) at the high concentration of 0.1%. Interestingly, the conditioners (containing no detergents) did not, at any concentrations studied, induce a Ca2+ influx. Plausibly, the unspecific Ca2+ influx induced by the higher concentrations of the children shampoo tested does not induce the smarting, stinging, and burning pain when exposed to the eyes as the regular shampoos do. TRPV1 has, in addition to chilli peppers, previously been shown to be regulated by commonly used compounds like ethanol (Trevisani et al., 2002) and camphor (Xu et al., 2005), and the new research areas involving TRPV1 will probably continue to grow. For instance, Bodo et al. (2005) showed that capsaicin administered on organ cultured human scalp hair follicles and outer root sheath keratinocytes expressing TRPV1 inhibited hair shaft elongation and proliferation, an interesting parallel to our results.
The corneal surface is the most highly innervated of all sense organs and the sensory nerves in the cornea and conjunctiva are predominantly free ending C-fiber nociceptors derived from the ophthalmic branch of the trigeminal nerve (MacIver and Tanelian, 1993; Rozsa and Beuerman, 1982). It is estimated that there are approximately 7000 nociceptors per mm2 in the human corneal epithelium, all ready to propagate impulses from painful stimuli (Muller et al., 2003) and eventually result in secondary effects of inflammation and possibly tissue damage if the stimulus is not removed (Geppetti and Holzer, 1996). Unfortunately, no in vitro alternatives to the Draize test include a nociceptive endpoint. The alternatives have proven to correlate well with the Draize rabbit eye test when testing severe irritants but failed to identify mild irritants (Debbasch et al., 2005). Alternative methods developed to replace the animal tests should be able to detect the whole spectrum of irritation, from corrosion to specific nociceptive responses. Maybe our neuronal cell model expressing TRPV1 could be an ultimate nociceptive endpoint. Previous studies by C. Belmonte and J. Gallar and colleagues show a correlation between irritation and different nociceptive responses (e.g., pain sensation, nerve activity, tear secretion) in conjunctiva and cornea, strengthening the hypothesis that a neuronal model measuring nociception mechanisms could be a completing assay for determination of eye irritation (Acosta et al., 2001a,b, 2004; Chen et al., 1995).
SLS dose-dependently induced a Ca2+ influx that could be blocked by capsazepine in the TRPV1-SH-SY5Y cells but no effects were seen in the native SH-SY5Y cells, suggesting that the TRPV1 receptor is the main respondent for the Ca2+ increase. However, we cannot exclude the involvement of other mechanisms of nociception, e.g., other voltage-sensitive Ca2+ channels (L- and N-type) (Reeve et al., 1994). Furthermore, the sensitivity of acid sensing ion channels (ASIDs) (Priest et al., 2006) or other TRP-channels for surfactants should be studied although TRPV1 seem to be the outermost chemically activated TRP-family member (TRP's reviewed in Ramsey et al., 2006).
In conclusion, SLS and hygiene products containing high concentration of SLS specifically activate the pain receptor TRPV1. Thus, TRPV1 activation might be a mediator of pain induced by detergent-hygiene products. Therefore, a TRPV1-expressing neuronal cell model could be useful as a mechanism-based component in an in vitro strategy for classification of eye irritating products.
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FUNDING |
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The Swedish Animal Welfare Agency; and the Swedish Fund for Research without Animal Experiments.
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ACKNOWLEDGMENTS |
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We thank Ms Astrid Blute for help with the immunofluorescence.
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