Skin is the largest organ and represents the body’s protective surface as the first contact site for topically applied substances. It plays a key role in controlling not only the penetration and distribution but also the metabolism of topically applied chemicals and is thus a first pass organ for penetrating substances.
This implies a need for safety assessment for all ingredients of cosmetic products and the need to employ appropriate models to test chemical ingredients for their potential to cause skin irritation, sensitization, as well as genotoxic damage
Animal testing was the method of choice for dermatotoxicological testing for decades. Since March 2013, animal testing for cosmetics was prohibited by the Seventh Amendment to the EU Cosmetics Directive, including skin irritation and genotoxicity. However, scientific community anticipated that it would take up to 10 years for the complete replacement of animal tests for skin sensitization, lung absorption or toxico kinetics (1). Moreover, the timing for the full replacement of animals use for carcinogenicity, repeat dose reproductive toxicology studies has not even been estimated (1).
The biotransformation activities in the skin mostly take place in the epidermis and are performed by keratinocytes (2, 3, 4).
The analysis of the expression and modulation of drug metabolizing enzymes in the skin is difficult, and for this reason, cultured primary keratinocytes have frequently been used to study epidermal drug metabolizing enzyme and drug metabolism.
To be a reliable model to mimic the in vivo situation, an in vitro epidermis model must retain xenobiotics metabolizing capabilities to a fully comparable level as in the skin. These should be comparable as expression of the different enzymes, as the magnitude of activities level as well as response to external stimulus and exposure to xenobiotics (e.g., enzymatic induction).
It has been demonstrated that primary human keratinocytes retain drug metabolizing activities in culture to comparable levels of that measured in skin sub-cellular preparations (S9 and microsomes) (5, 6).
Noteworthy, primary human keratinocytes showed to retain closer to skin preparations non-cytochrome P450 enzymatic activities, than other models. In particular for cyclooxygenases (COX), NADPH quinone reductases (NQR), UGT, NAT and GST activities (5, 6). This is a very important characteristic, in fact, it is reported that the levels of skin expression and activities of cytochrome P450 enzymes are very low, often not detectable. Therefore the non-cytochrome P450 enzymes are in the skin the major responsibilities of xenobiotics metabolism. For example, the cutaneous COX activities are important in the oxidative metabolism in the skin, since COX can metabolize many lipophilic xenobiotics with a low redox potential including many amines (7). A comparison of reported enzymatic activities measured in human primary keratinocytes as compared to the skin has been reviewed (6) and relevant data are summarized in Table 1.
It has been noted that levels of expression of drug metabolizing enzymes in the skin show a notable inter-individual variability (8). For example, it has been reported variability of 98 fold in the skin expression of CYP3A4/3A5 (8, 9, 10). Moreover, in some individual have been found skin levels expression of CYP3A4/3A5 comparable to those observed in liver cells (8). The large variability of expression of drug metabolizing enzymes is likely to depend on a number of factors including differences in assay culture conditions, genetic background, and lifestyle. It is, however, important to note that primary keratinocytes in culture retain these inter-individual differences making them a representative and useful model also for the evaluation of the relevant of inter-individual variability in xenobiotics metabolism.
The expression levels of drug metabolizing enzymes are regulated by a number of nuclear receptors (11, 12, 13, 14, 15, 16) and can be induced by the exposure to various xenobiotics. It has been reported that many classical inducers are also active in cultured primary keratinocytes (17). Moreover, the magnitude of induction levels of CYP enzymes, UGT, and GST have been observed to be similar if not higher in cultured keratinocytes as those observed in cultured human hepatocytes. Showing also a notable inter-individual variability in the response (8) likely due to a notable difference in the expression of key nuclear receptors.
Therefore, primary human keratinocytes are a relevant and simple model for the study of enzymatic induction in skin; this is an important area of investigation as a strong induction of some low or not expressed enzymes may result in a dramatic change of the overall drug metabolizing competency with potential undesirable physiological and toxicological outcomes.
For the use of primary keratinocytes for drug metabolism studies, it is important to consider that maturation, cryopreservation and culture methods used have notable effects on the activities of the cells. Keratinocytes drug metabolizing capabilities are not significantly altered by cryopreservation nor by secondary culturing as compared to fresh cells (5).
Keratinocytes in vivo undergo a continuous process of self-renewal and the progressively differentiated towards the outermost layer of the skin tissue. Mature fully differentiated keratinocytes show full drug metabolizing capabilities. Therefore it is important to use differentiate cells for drug metabolism studies aimed to be relevant to mimic the in vivo conditions.
In vitro, differentiation of cultured keratinocytes is typically induced via three approaches.
- The first allows the cells to become confluent in media containing high calcium. Thus, differentiation is initiated by both cell-cell contact and increased calcium levels.
- The second approach is to induce the cells to differentiate by suspending the proliferating cells in a matrix, typically methyl cellulose. It is thought that the loss of adhesion is the event that induces differentiation (18).
- The third approach, the organotypic method, allows for the formation of the multilayers of the epithelium in the culture dish (19). In this system, the keratinocytes form layers representative of each stage of differentiation, much like that observed in vivo. A critical feature of this approach is that the epithelium is floating on a collagen gel at the air-liquid
Each of these three approaches has unique advantages and disadvantages. In the first and second approaches is that differentiation can be observed in a relatively short period of time. The entire course of differentiation occurs within approximately 2–6 days in the calcium-calcium switch method and within 24 h with the suspension-induced differentiation protocol. In contrast, a well-developed cornified layer indicative of full differentiation is typically achieved by eight days of organotypic culture. However, in accelerated differentiation (approaches 1 and 2) the cells are subjected to more culture-induced stress. For example, it is thought that the highly confluent nature of the cells as they differentiate accelerate stress-induced senescence (20).
The differentiation status of keratinocytes has an impact on the expression and activities of nuclear receptors that regulate the expression and inducibility of many drug metabolizing enzymes (e.g., GR, VDR, and AHR). The increase of differentiation correlates with a decrease of GR and VDR expression, while it correlates with the increase of AHR receptor levels. Thus, this could result in a different profile of response to inducers of keratinocytes used at different levels of maturation and differences between the drug metabolizing enzymes in the magnitude of the response. This area needs to be investigated in more in details as a systemic comparative study on this subject has not been reported yet.
In addition to maturation, also culturing conditions have a relevant role in the performance of different studies.
In cultured primary human keratinocytes, has been reported that drug metabolism activities (all those investigated) decrease at the post-confluent condition and are at the highest level in pre-confluent conditions (5). This suggests that pre-confluent culturing conditions are ideal for drug metabolism, drug profiling, and clearance studies.
On the contrary, primary keratinocytes showed a higher response to inducer in post-confluent cultures for AHR regulated enzymes while no differences have been observed for drug metabolizing enzymes regulated by other nuclear receptors.
In conclusion, primary human keratinocytes show similar to in vivo skin drug metabolism capabilities, their xenobiotics metabolism competency is not altered by sub-culture nor cryopreservation procedure. In addition, cultured primary human keratinocytes are responsive to several known chemical enzyme inducers.
Thus, primary human keratinocytes represent a simple and suitable in vitro system for the study of the biotransformation and cytotoxicity of topically applied compounds. It compared well with other in vitro model such as keratinocytes-derived reconstituted skin model.
- Adler S., Basketter D.,Creton S., Pelkonen O., van Benthem J., Zuang V., Andersen K.E., Angers-Laustau A., Aptula A., Bal-Prince A., Benfenati E., Bernauer U., Bessems J., Bois F.Y., Boobis A., Brandon E., Bremer S., Broschard T., Casati S., Coecke S., Corvi R., Cronin M., Daston G., Dekant W., Felter S., Grignard E., Gundert-Remy U., heinonen T., Kimber I., Kleinjans J., Kamulainen H., Kreiling R., Kreysa J., Leite S.B., Loizou G., Maxwell G., Mazzatorta P., Munn S., Pfuhler S., Phrakonkham P., Piersma A., Porth A., Prieto P., Repetto G., Rogiers V., Schoeters G., Schwarz M., Serafimova R., Tahti H., Testai E., van Delft J., van Loveren H., Vinken M., Worth A. and Zaldivar J.-M. Toxicol. 2011. 85: 367-485
- D.R. Bikers, T. Dutta-Choudhury and H. Mukhtar. Pharmacol. 1982. 21: 239-247.
- D.R. Bikers, C. L. Marcelo, T. Dutta-Choudhury and H. Mukhtar. J.Pharmacol. Exp.Ther. 1982. 223: 163-168
- W. A. Khan, S. S. Park, H. V. Gelboin, D. R. Bikers and H. Mukhtar. Pharmacol. Exp. Ther. 1989. 249: 921-927
- B. Hirel, C. Chesne, J. P. Pailheret and A. Guillouzo. Toxicol. In Vitro. 1995. 1: 49-56
- Oesch, E. Fabian, K. Guth and R. Landsiedel. Arch. Toxicol. 2014. 88: 2135-2190.
- Oesch-Bartlomowicz and F. Oesch. B. Testa and H. van de Waterbeemd eds. Comprensive Medical Chemistry. Elsevier, Oxford. 2007. 193-214.
- I. Swanson. Chemico-Biol Interact. 2004. 149:69-79.
- R. Andersen, F. M. Farin and C. J. Omiecinski. DNA Cell Biol. 2001. 17: 231-238.
- Janmohamed, D. Holler, I. R. Philips and E.A. Shepard. Biochem. Pharmacol. 2001. 62: 777-786.
- D. W. Nebert, T. P. Dalton, A. B. Okey and F. J. Gonzalez. J. Biol. Chem. 2004. 279: 23847-23850.
- J. M. Maglich, C. M. Stoltz, B. Goodwin, D. Hawkins-Brown, J. T. Moore and S. A. Kliewer. Mol. Pharmacol. 2002. 62: 638-646.
- J. M. Maglich, D. J. Parks, L. B. Moore, J. L. Collins, B. Goodwin, A. N. Billin, C. M. Stoltz, S. A. Kliewer, M. H. Lambert, T. M. Wilson and J. T. Moore. J. Biol. Chem. 2003. 278:17277-17283.
- J. M. Pascussi, S. Gerbal-Chaloin, L. Drocourt, P. Maurel and M. J. Vilarem. Biochim. Biophys. Acta. 2003. 1619: 243-253.
- Y. Chen, S. S. Fergusson, M. Negishi and J. A. Goldstein. J. Pharmacol. Exp. Ther. 2004. 308:495-501.
- S. Gerbil-Charloin, J. M. Pascussi, L. Pichard-Garcia, M. Daujat, Fwaechter, J. M. Fabre, N. Carrere and P. Maurel. Drug Metab. Dispos. 2001. 29: 242-251.
- l. M. Baron, D. Holler, R. Schiffer, S. Frankenberg, M. Neis, H. F. Merk and F. K. Jugert. Invest. Dermatol. 2001. 116: 541-548.
- F. M. Watt, P. W. Jordan and C. H. O’Neill. Natl. Acad. Sci. U.S.A. 1988. 85: 5576-5580.
- A. Li, N. Pouliot, R. Redvers and P. Kaur. Clin. Invest. 2004. 113: 390-400.
- S. S. Ray and H. I. Swanson. Appl. Pharmacol. 2003. 192: 131-145.