Most people use cosmetics every day. For instance,

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1 DERMATOLOGY FUMITAKA FUJITA Central Research Laboratories, Mandom Corp Osaka, Osaka , Japan Transient receptor potential channels in sensory irritation TRP channels as tools for the predictive identifi cation of sensory irritation Sensory irritation, such as stinging, pricking or burning sensations, can be occasionally caused by skin Abstractcosmetics. Since skin cosmetics are used daily and left on the skin surface for an extended period of time after application, any associated sensory irritation can be unpleasant for consumers. However, the mechanisms of sensory irritation caused by chemical stimuli on the skin surface are still unknown. Recently, it has been demonstrated that Transient Receptor Potential (TRP) channels are described to potentially have important roles in sensing nociceptive stimuli. We determined that parabens and alkali agents cause TRPA1, a nociceptive sensor, activation to establish an alternative evaluation method for sensory irritation from cosmetics. INTRODUCTION Most people use cosmetics every day. For instance, each day, people may use lipstick, skin toner, foundation, shampoo, body soap, perfume, shaving foam, etc. Potentially, the use of cosmetics can lead to skin irritation. Skin irritation can be classified roughly into two phenomena: skin inflammation and sensory irritation. Alternative methods for evaluation of skin inflammation are very advanced, as several reports involving 3D skin or cultured cells have been published (1, 2). Although a few reports have been published regarding alternative tests for sensory irritation (3, 4), we were not aware of a useful alternative test for sensory irritation. Knowledge about sensory irritation has accumulated recently and as a result, sensory irritation by cosmetics was found to be rare. However, many consumers seem to have given up using cosmetics or have started using other cosmetics because of sensory irritation. When a consumer experiences stinging or burning from using skin cosmetics, how does he/she feel? If it happens the first time she uses that kind of skin cosmetic, he/she may never use it again. We must see it as a lost chance at making one of our customers more beautiful. On the other hand, for those of us who work in cosmetics companies, we often tend to think that this sensory irritation must be caused by the consumer s damaged skin, because the product has passed our safety test. If we ignore the problem, however, the consumer will probably have a bad image of all of our products. This was our motivation for developing an evaluation method for sensory irritation. Here I introduce the results of questions posed to young Japanese women (N=312). 21

2 The first question was What kind of skin trouble have you had? For each kind of cosmetic, irritation was answered by 75 percent of the subjects and the most frequent answer (skin redness: 56 percent, skin eruption: 37 percent, skin roughness: 33 percent). Therefore, the second question was What kind of irritation have you experienced? Stinging or burning was answered by 65 percent of the subjects and the most frequent answer (itching: 35 percent, skin flushing: 22 percent). Consequently, we think that female consumers often recognize skin troubles with sensations such as stinging or burning. These results indicate that we should pay more attention to sensory irritation. However, ten years ago, the mechanisms of sensory irritation were not well understood. Recently, it has become clearer how individuals feel sensory irritation at the molecular level. thermosensation has made rapid progress since TRPV1 was found to be activated by noxious high temperature stimuli (6, 7). Now, we know that temperatures ranging from noxious heat to noxious cold activate several members of the thermosensitive TRP channels. In particular, TRPV1 and TRPM8 are well known in the cosmetic field (8, 9). Of these nine thermosensitive TRP channels, six are thought to be expressed in sensory neurons and to be involved in nociception or pain relief. They represent sensitivity to a range of temperatures from noxious heat (TRPV1, TRPV2) and warmth (TRPV3, V4) and cold to innocuous cool (TRPM8, A1) (Figure 1). MECHANISM OF SENSORY IRRITATION How do individuals feel sensory irritation? As we know, nociceptive stimulation to the skin, such as cold, hot or chemical stimulation, is detected at the peripheral nerve endings and transmitted to our brain through the nerves and spinal cord as electrical signals. Recently, what happens in the skin and sensory nerves when we feel pain has become clearer. In the membrane of sensory neurons, there are various receptors for nociceptive stimuli and temperature. In many cases, the receptor is a cation channel. When the ion channel is activated by chemicals or temperature, cations like sodium and calcium flow into the cell, resulting in neural excitation. This is the mechanism by which chemical and temperature stimuli are converted to electrical signals. These ion channels are embedded in the plasma membrane, and electrochemical gradients exist across the lipid bilayer. Inside the cell, the potassium concentration is higher than outside the cell, and outside the cell, the sodium and calcium concentrations are higher than inside the cell. Thus, when a cationic channel opens, cations diffuse rapidly down their electrochemical gradients selectively or non-selectively. Many kinds of ion channels are related to our sensations. Of them, we focused on one group of ion channels named Transient Receptor potential (TRP) channels, which have important roles in thermosensation. THERMOSENSITIVE TRP CHANNELS TRP channels were originally identified in Drosophila and found to be involved in phototransduction (5). TRP was deficient in a Drosophila mutant that exhibited abnormal transient responsiveness to continuous light. These ion channels are thought to have intracellular amino and carboxyl termini with six putative transmembrane segments and are thought to work as non-selective cation channels with high Ca 2+ permeability. Many invertebrate and vertebrate homologues of the Drosophila TRP channels have been cloned. They consist of a large superfamily, which has the following seven subfamilies: V, M, A, N, P, ML and C and they have a key role in sensing various stimuli. Some of these TRP channels are thought to sense chemicals that cause sensory irritation or temperature. In mammals, nine thermosensitive TRP channels are thought to be involved in the perception of ambient temperature. Our understanding of the molecular mechanisms of Figure 1. Thermosensitive-TRP channels expressed in sensory neurons. TRPV1 AND TRPV2 Capsaicin, a main component of hot chili pepper, activates TRPV1, as identified by Julius and colleagues (6). TRPV1 was the first identified thermally gated cation channel activated by noxious heat (>42 C) and is expressed in small-diameter sensory neurons (6, 7). Moreover, TRPV1 is a promiscuous channel that is activated by a wide range of agonists, such as capsaicin, low ph, ethanol, camphor, allicin, gingerol, shogaol, piperine, etc. (7, 10). Since various inflammatory mediators sensitize TRPV1 indirectly via phosphorylation, TRPV1 is thought to have an important role in inflammatory pain hypersensitivity (11). Therefore, in the cosmetic field, TRPV1 was reported as a sensor for detection of eye irritants (8). Residual noxious heat sensation above 52 C in TRPV1 knockout mice led to the discovery of the second thermo TRP, TRPV2 (12). TRPV2 is localized in medium- to largediameter sensory neurons and activated by high heat and mechanical stretch (13). However a direct role of TRPV2 in pain sensation remains elusive, as TRPV2 null mice showed no evident deficits in thermal and pain sensation (14). TRPV3 AND TRPV4 TRPV3 is activated by warm temperature with a threshold of C. Since camphor, carvacrol, eugenol and thymol also activate TRPV3, it is thought to be a molecular target of plant-derived skin sensitizers (16). TRPV3 is prominently expressed by keratinocytes within the skin. Therefore, in keratinocytes, TRPV3 transmits temperature information to sensory neurons via ATP (17). TRPV3 is also required for the 22 Household and Personal Care Today - Vol. 7 nr. 4 October/December 2012

3 formation of the skin barrier by regulating the activities of transglutaminases (18). TRPV4 is identified as a channel highly sensitive to changes in extracellular osmolarity (19). Warm temperature (27-35 C) also activates TRPV4 (20). TRPV4 is mainly expressed by keratinocytes in the skin, and physiological skin temperatures play important roles in skin barrier homeostasis through TRPV4 activation (21). These previous reports show that these two thermosensitive TRP channels are thought to have important roles in the maintenance of skin barrier function. TRPM8 AND TRPA1 Soon after the discovery of TRPV1, a cold-sensitive TRP channel, TRPM8 was cloned (22). TRPM8 is activated by menthol, which produces a cooling sensation on the skin surface, and menthol derivatives (9, 22). The in vivo expression pattern of TRPM8 appears to be approximately 10 percent small-diameter sensory neurons without TRPV1 co-expression (22). Moreover, the majority of the studies with TRPM8 antibodies did not detect co-localization of this channel with TRPV1. TRPA1 was identified as a protein overexpressed in liposarcoma cell lines (23). Recently, it became clear that TRPA1 has an important role in nociception and is activated by isothiocyanate or thiosulfinate compounds like allyl isothiocyanate, the main ingredient of mustard oil or horseradish, and cinnamaldehyde, the main ingredient of cinnamon (24, 25). There are increasing numbers of reports about the stimulus for TRPA1 activation. Although TRPA1 has an activation threshold approaching that of cold pain (<17 C), there is a difference of opinion as to whether TRPA1 actually has a function as a cold detector (25). Since menthol also activates TRPA1 in humans, high doses of this compound are thought to cause pain sensation on the skin surface (26). Consequently, we chose to investigate thermosensitive TRP channels as tools for the predictive identification of sensory irritation. plasma membrane. For the patch clamp method, patch electrodes were made from pieces of fine glass tubing and filled with conducting solution. A Whole-cell clamp, which can record the electrical behaviour of the cell, was often chosen. The tip of the patch electrode can be attached directly to the plasma membrane. Current changes can be recorded while the cell is being exposed to a chemical or a change in temperature. Among the thermosensitive TRP channels, we focused on TRPA1, a nociceptive sensor. We started this project about seven years ago. At that time, the molecular mechanisms of sensory irritation caused by parabens and alkali agents, which are known as irritants contained in cosmetic products, were not understood. Therefore, we evaluated these irritants with thermosensitve TRP channels (28, 29). PARABENS We first examined the effects of methyl paraben. This antibacterial agent is considered to be safe as used in cosmetic formulation. However, some reports show methyl paraben causes sensory irritation on skin surface of sensitive volunteers in Japan (30). Using a Ca 2+ -imaging method, we found that it increased cytosolic Ca 2+. To address whether the methyl paraben-induced increases in Ca 2+ were specific to TRPA1, we examined the effects of 1 mm methyl paraben on Ca 2+ in HEK293 cells expressing other TRP channels, TRPV1, TRPV2, TRPV3, TRPV4 or TRPM8. Methyl paraben caused a Ca 2+ increase in HEK293 cells overexpressing TRPA1, but not in cells overexpressing the others, suggesting a selective action of CA 2+ -IMAGING AND PATCH CLAMP ANALYSIS In the study of thermosensitive TRP channels, we usually use two methods. One is a calcium imaging method. In short, it is a method of observing changes in cellular calcium by using fluorescent material. The calcium imaging method is thought to be easy for identifying chemicals that have an effect on non-selective cation channels. It is conducted as follows. First, the fluorescent indicator is made to be taken into the cells. Next, we mount the cells that overexpress the target ion channels on a coverslip in a chamber. Then, we can measure the fluorescence signals while the material is being applied. Changes in intracellular calcium can be measured as a ratio of the signals evoked by two excitation wavelengths, 380 nm and 340 nm. Human embryonic kidneyderived (HEK293) cells are reported as useful cells for studies of the target TRP channel without native other TRP channels. Therefore, HEK293 cells overexpressing TRP channels with lipofectamine are used in our studies. Data from this method showed good correlation with sensory irritation scores in vivo using anti-bacterial agents and polyols (27). To evaluate TRP channels activation directly, we performed patch clamp recording. This method can measure current flow across the Household and Personal Care Today - Vol. 7 nr. 4 October/December

4 methyl paraben on TRPA1 channels. Moreover, all of the parabens examined, including methyl paraben, ethyl paraben, propyl paraben and butyl paraben, had an ability to increase Ca 2+ in HEK293 cells overexpressing TRPA1 channels. These results suggest that benzoate, a common structural feature of the parabens, might be involved in the actions of the parabens on TRPA1 channels. To examine more directly whether methyl paraben activates TRPA1 channels, we performed patchclamp experiments in HEK293 cells overexpressing TRPA1 channels. Methyl paraben evoked inward currents that exhibited an EC 50 of 4.4 mm in HEK293 cells overexpressing TRPA1 channels. Furthermore, 10 µm ruthenium red and 5 mm camphor, as inhibitors of TRPA1, caused almost complete inhibition of methyl paraben-induced TRPA1 currents. These results indicate that methyl paraben causes a stinging sensation through activation of TRPA1 channels. ALKALI AGENTS High amounts of alkali caused sensory irritation with the use of hair dye near the head. Therefore, we focused on alkali agents as irritants contained in hair dye. We first examined whether alkali ph could activate TRPA1 in vitro using a Ca 2+ - imaging method. Not only ph 8.5 alkali solution but also 5 mm ammonium chloride, which caused an increase in intracellular ph through the binding of ammonia with protons in the cytosol, induced an increase in intracellular Ca 2+ concentration in cells expressing TRPA1. Moreover, this activation was caused only in the cells expressing TRPA1 among other TRP channels expressed in sensory nerves. These results suggested that intracellular alkalization was important for TRPA1 activation. To confirm whether intracellular alkali ph caused TRPA1 activation, we performed inside-out patch clamp experiments in cells expressing TRPA1. Increasing the bath ph to 8.0 or 8.5 increased the channel opening in a ph-dependent manner, indicating the existence of alkalization-induced activation of the ion channels in the patch membrane. These results suggest that TRPA1 activation by intracellular alkaline ph occurs at a single-channel level. To create low-sensory irritation hair-dye, we looked for a way to block intracellular alkalization. We found that carbonate ion can block intracellular alkalization. Moreover, in a model hair dye, sensory irritation was decreased by carbonate ion. At last, we could create low-sensory irritation hair dye using knowledge about TRP channels. The progress in our evaluation technology for sensory irritation will make it possible for us to develop cosmetic products with reduced stinging sensation and controlled cool sensation without using human studies. Recently, we found a novel TRPA1 antagonist and have developed cooling cosmetics with it. In the future, many formulation technologies will be developed based on this knowledge of TRP channels. REFERENCES AND NOTES 1. J.H. Fentem, D. Briggs et al., Toxicol In Vitro, 15, pp (2001). 2. J.H. Fentem, M. Chamberlain et al., Altern Lab Anim., 32, pp (2004). 3. P.J. Frosch, A.M. Klingman, J Soc Cosmet Chem., 28, pp (1977). 4. B. Querleux, K. Dauchot et al., Skin Res Technol., 14, pp (2008). 5. C. Montell, G.M. Rubin, Neuron, 2, pp (1989). 6. M.J. Caterina, M.A. Schumacher et al., Nature, 389, pp (1998). 7. M. Tominaga, M.J. Caterina, J. Neurobiol., 61, pp (2004). 8. J. Lilja, H. Lindegren et al., Toxicol Sci., 99, pp (2007). 9. H.J. Behrendt, T. Germann et al., Br J Pharmacol., 141, pp (2004). 10. L.J. Macpherson, B.H. Geierstanger et al., Curr Biol., 15, pp (2005). 11. D. Julius, A.I. Basbaum, Nature, 413, pp (2001). 12. M.J. Caterina, T.A. Rosen et al., Nature, 398, pp (1999). 13. K. Muraki, Y. Iwata et al, Circ Res, 93, pp (2003). 14. U. Park, N. Vastani et al., J Neurosci., 32, pp (2011). 15. A.M. Peier, A.J. Reeve et al., Science, 296, pp (2002). 16. H. Xu, M. Delling et al., Nat Neurosci., 9, pp (2006). 17. S. Mandadi, T. Sokabe et al., Pflugers Arch., 458, pp (2009). 18. X. Cheng, J. Jin et al., Cell, 141, pp (2010). 19. R. Strotmann, C. Harteneck et al., Nat Cell Biol., 2, pp (2000). 20. A.D. Güler, H. Lee et al., J Neurosci., 22, pp (2002). 21. N. Kida, T. Sokabe et al., Pflugers Arch., 463, pp (2012). 22. D.D. McKemy, W.M. Neuhausser et al., Nature, 416, pp (2002). 23. D. Jaquemar, T. Schenker et al., J Biol Chem., 274, pp (1999). 24. G.M. Story, A.M. Peier et al., Cell, 112, pp (2003). 25. D.M. Bautista, S.E. Jordt et al., Cell., 124, pp (2006). 26. B. Xiao, A.E. Dubin et al., J Neurosci., 28, pp (2008). 27. F. Fujita, S. Iwashita, Proceedings of the 26 th Congress of The International Federation of Cosmetics Chemists, Buenos Aires September, p.48 (2010). 28. F. Fujita, Proceeding of 6th World Congress on Alternatives & Animal Use in the Life Sicences, Tokyo August, p.115 (2007). 29. F. Fujita, M. Tominaga et al., Proceedings of the 9th Asian Societies of Cosmetic Scientists, Yokohama 2-4 March, pp (2009). 30. T. Sone, H. Yamada et al., J Jpn Cosmet Sci Soci., 14, pp (1990). 24 Household and Personal Care Today - Vol. 7 nr. 4 October/December 2012

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