Cell Biol Toxicol (2011) 27:69–81 not alter the cells' properties, which illustrate the resistance of keratinocytes as compared to other cell types tested in this respect. Ultrastructural examination revealed stress-related granules in fluoride-treated cells. In addition, PCR confirmed the induction of HSC70, a stress-related protein, and immunolabeling revealed its presence in cytoplasmic foci (Fig. 4). Presence of stress indicators, but absence of apoptosis and necrosis features, as well as ongoing cell proliferation in stress conditions, might suggest that withdrawal of fluoride restores the normal phenotype of HaCaT cells immediately. However, we found that the fluoride-related keratin expression pattern was maintained after the removal of fluoride and further cell growth. This would suggest that fluoride-induced changes not only bring about stress but they also exert in addition an independent effect on cells. In this sense, the changes of the keratin pattern may not represent a stress effect. Concerning effects of keratin misexpression in vivo, deficient expression of single keratins is generally compensated by other keratins. However, for the case of the double KO k18-19, severe embryonal disturbances have been observed (Arin 2009), and mutations in keratins k1-10 as well as k13 resulted in distinct phenotypes of adult human beings (Arin 2009), in particular when exposed to environ- mental stress. If keratin gene expression would be disturbed by fluoride in vivo in a similar way as described here, it is conceivable that cell interactions could be altered. In this way, epithelia formation might be compromised so that organs like intestine, kidney, and lung exhibit functional disturbances. In addition, changes in cell metabolism would be expected that could further contribute to reduced organ function. The observed effects on keratinocytes occurred at concentrations that were considerably higher than concentrations affecting osteoblasts, odontoblasts, and especially ameloblasts. These differences have repeatedly been observed. Considering tissue growth and organ development as a whole, the most striking effects of fluoride occur on the formation of mineralized tissue. Effects on epithelia during development would thus only take place if the general development and tissue formation were totally deranged with catastrophic consequences for the organism as a whole. Therefore, consequences of fluoride as described here are likely to 79 concern renewing epithelia of the adult organism. Examples of possible local fluoride exposures are lung epithelia through the air, kidney epithelia due to secreted fluoride, or mucosal epithelia due to ingested fluoride (Whitford 1990; Gharzouli et al. 2000). The mechanism of fluoride action is unknown; the wide variations of effective fluoride concentrations suggest that different cell types react in a different way. It has been proposed that cells may occupy a set of physiological states, biological attractors (Kitano 2004). These states are discrete, because the total set of cell products must accommodate metabolic func- tions, gene regulation, cell communication etc., and it is likely that only few physiological states can match all requirements. Robustness towards environmental changes is favored if cells may switch between several states. Presence of fluoride might force cells to occupy states different from control cells, whereby the synthesis of many cell products may be altered. It is conceivable that the effective concentration depends on the cell type; therefore, this hypothesis is consistent with grossly different fluoride levels affecting different cell types. It seems likely that cells of epithelia have been selected during evolution to support variable ranges of external factors, whereas less pressure is exerted on mesenchymal cells, not forming extracorporal surfaces. Acknowledgements We thank Dr. N.E. Fusenig for kindly providing HaCaT cell line to our laboratory. We thank Mrs. Martine Oboeuf for EM technique. This work was supported by a Convention Industrielle de Formation par la Recherche (CIFRE) grant in collaboration with Sanofi-Aventis OTC directed by P. Ben-Soussen (deceased), medical director, Sanofi-Aventis OTC and especially C. Gal-Jallat, scientific interlocutor for Sanofi-Aventis OTC. In memoriam to Dr. Isabelle Fernandes, who was a great friend, a great colleague, and a great researcher. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Arin MJ. The molecular basis of human keratin disorders. Hum Genet. 2009;125(4):355–73. Bevilacqua E, Wang X, Majumder M, Gaccioli F, Yuan CL, et al. eIF2alpha phosphorylation tips the balance to apoptosis