Reconstructed epidermis and full-thickness skin for absorption testing: influence of the vehicles used on steroid permeation.

A protocol for percutaneous absorption studies has been validated, based on the use of reconstructed human epidermis (RHE) and aqueous solutions of test substances. However, it is often the case that it is more-complex formulations of drugs or chemicals which will make contact with the skin surface. To investigate whether RHE and the reconstructed full-thickness skin model (FT-model) can be used to predict uptake from formulations, we compared the permeation of hydrocortisone and testosterone when applied in emulsion form and as a solution containing the penetration enhancer, ethanol. Human and pig skin and a non-cornified alveolar model served as references. The results were compared with steroid release from the formulations. The permeation rates of the steroids were ranked as: alveolar model >> RHE > FT-model, pig skin > human skin. In accordance with the rapid hydrocortisone release from the formulations, the permeation rates of this steroid exceeded those of testosterone. Only minor differences were observed when comparing the testosterone formulations, in terms of release and permeation. However, the ranking of the permeation of the hydrocortisone formulations was: solution > w/o emulsion > o/w emulsion, which permitted the elucidation of penetration enhancing effects, which is not possible with drug release studies. Differences in penetration were most obvious with native skin and reconstructed tissues, which exhibited a well-developed penetration barrier. In conclusion, RHE and skin preparations may be useful in the development of topical dermatics, and in the framework of hazard analysis of toxic compounds and their various formulations.

The use of reconstructed human epidermis for skin absorption testing: Results of the validation study.

A formal validation study was performed, in order to investigate whether the commercially-available reconstructed human epidermis (RHE) models, EPISKIN, EpiDerm and SkinEthic, are suitable for in vitro skin absorption testing. The skin types currently recommended in the OECD Test Guideline 428, namely, ex vivo human epidermis and pig skin, were used as references. Based on the promising outcome of the prevalidation study, the panel of test substances was enlarged to nine substances, covering a wider spectrum of physicochemical properties. The substances were tested under both infinite-dose and finite-dose conditions, in ten laboratories, under strictly controlled conditions. The data were subjected to independent statistical analyses. Intra-laboratory and inter-laboratory variability contributed almost equally to the total variability, which was in the same range as that in preceding studies. In general, permeation of the RHE models exceeded that of human epidermis and pig skin (the SkinEthic RHE was found to be the most permeable), yet the ranking of substance permeation through the three tested RHE models and the pig skin reflected the permeation through human epidermis. In addition, both infinite-dose and finite-dose experiments are feasible with RHE models. The RHE models did not show the expected significantly better reproducibility, as compared to excised skin, despite a tendency toward lower variability of the data. Importantly, however, the permeation data showed a sufficient correlation between all the preparations examined. Thus, the RHE models, EPISKIN, EpiDerm and SkinEthic, are appropriate alternatives to human and pig skin, for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions.

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study.

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.

The estrogen-related receptor alpha (ERRalpha) functions in PPARgamma coactivator 1alpha (PGC-1alpha)-induced mitochondrial biogenesis.

Estrogen-related receptor alpha (ERRalpha) is one of the first orphan nuclear receptors to be identified, yet its physiological functions are still unclear. We show here that ERRalpha is an effector of the transcriptional coactivator PGC-1alpha [peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha], and that it regulates the expression of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Inhibition of ERRalpha compromises the ability of PGC-1alpha to induce the expression of genes encoding mitochondrial proteins and to increase mitochondrial DNA content. A constitutively active form of ERRalpha is sufficient to elicit both responses. ERRalpha binding sites are present in the transcriptional control regions of ERRalpha/PGC-1alpha-induced genes and contribute to the transcriptional response to PGC-1alpha. The ERRalpha-regulated genes described here have been reported to be expressed at reduced levels in humans that are insulin-resistant. Thus, changes in ERRalpha activity could be linked to pathological changes in metabolic disease, such as diabetes.

Endurance training in humans leads to fiber type-specific increases in levels of peroxisome proliferator-activated receptor-gamma coactivator-1 and peroxisome proliferator-activated receptor-alpha in skeletal muscle.

The peroxisome proliferator-activated receptor (PPAR)-gamma coactivator-1 (PGC-1) can induce mitochondria biogenesis and has been implicated in the development of oxidative type I muscle fibers. The PPAR isoforms alpha, beta/delta, and gamma control the transcription of genes involved in fatty acid and glucose metabolism. As endurance training increases skeletal muscle mitochondria and type I fiber content and fatty acid oxidative capacity, our aim was to determine whether these increases could be mediated by possible effects on PGC-1 or PPAR-alpha, -beta/delta, and -gamma. Seven healthy men performed 6 weeks of endurance training and the expression levels of PGC-1 and PPAR-alpha, -beta/delta, and -gamma mRNA as well as the fiber type distribution of the PGC-1 and PPAR-alpha proteins were measured in biopsies from their vastus lateralis muscle. PGC-1 and PPAR-alpha mRNA expression increased by 2.7- and 2.2-fold (P < 0.01), respectively, after endurance training. PGC-1 expression was 2.2- and 6-fold greater in the type IIa than in the type I and IIx fibers, respectively. It increased by 2.8-fold in the type IIa fibers and by 1.5-fold in both the type I and IIx fibers after endurance training (P < 0.015). PPAR-alpha was 1.9-fold greater in type I than in the II fibers and increased by 3.0-fold and 1.5-fold in these respective fibers after endurance training (P < 0.001). The increases in PGC-1 and PPAR-alpha levels reported in this study may play an important role in the changes in muscle mitochondria content, oxidative phenotype, and sensitivity to insulin known to be induced by endurance training.

The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor estrogen-related receptor alpha (ERRalpha).

The estrogen-related receptor alpha (ERRalpha) is one of the first orphan nuclear receptors identified. Still, we know little about the mechanisms that regulate its expression and its activity. In this study, we show that the transcriptional coactivator PGC-1, which is implicated in the control of energy metabolism, regulates ERRalpha at two levels. First, PGC-1 induces the expression of ERRalpha. Consistent with this induction, levels of ERRalpha mRNA in vivo are highest in PGC-1 expressing tissues, such as heart, kidney, and muscle, and up-regulated in response to signals that induce PGC-1, such as exposure to cold. Second, PGC-1 interacts physically with ERRalpha and enables it to activate transcription. Strikingly, we find that PGC-1 converts ERRalpha from a factor with little or no transcriptional activity to a potent regulator of gene expression, suggesting that ERRalpha is not a constitutively active nuclear receptor but rather one that is regulated by protein ligands, such as PGC-1. Our findings suggest that the two proteins act in a common pathway to regulate processes relating to energy metabolism. In support of this hypothesis, adenovirus-mediated delivery of small interfering RNA for ERRalpha, or of PGC-1 mutants that interact selectively with different types of nuclear receptors, shows that PGC-1 can induce the fatty acid oxidation enzyme MCAD (medium-chain acyl-coenzyme A dehydrogenase) in an ERRalpha-dependent manner.

The PGC-1-related protein PERC is a selective coactivator of estrogen receptor alpha.

Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) is a tissue-specific coactivator that enhances the activity of many nuclear receptors and coordinates transcriptional programs important for energy metabolism. We describe here a novel PGC-1-related coactivator that is expressed in a similar tissue-specific manner as PGC-1, with the highest levels in heart and skeletal muscle. In contrast to PGC-1, the new coactivator shows high receptor specificity. It enhances potently the activity of estrogen receptor (ER) alpha, while having only small effects on other receptors. Because of its nuclear receptor selectivity, we have termed the new protein PERC (PGC-1 related Estrogen Receptor Coactivator). We show here that the coactivation function of PERC relies on a bipartite transcriptional activation domain and two LXXLL motifs that interact with the AF2 domain of ERalpha in an estrogen-dependent manner. PERC and PGC-1 are likely to have different functions in ER signaling. Whereas PERC acts selectively on ERalpha and not on the second estrogen receptor ERbeta, PGC-1 coactivates strongly both ERs. Moreover, PERC and PGC-1 show distinct preferences for enhancing ERalpha in different promoter contexts. Finally, PERC enhances the ERalpha-mediated response to the partial agonist tamoxifen, while PGC-1 modestly represses it. The two coactivators are likely to mediate distinct, tissue-specific responses to estrogens.