Carsten Goebel

Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I)

Abstract:  The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first‐pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic‐metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S‐transferase, UDP‐glucuronosyltransferase and N‐acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI‐200), immortalized keratinocyte‐based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI‐200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.

Categorization of chemicals according to their relative human skin sensitizing potency.

Although adoption of skin sensitization in vivo assays for hazard identification is likely to be successful in the next few years, this does not replace their use in potency prediction. Notably, measurement of potency of skin sensitizers in the local lymph node assay has been important. However, this local lymph node assay potency measure has not been formally assessed against a range of substances of known human sensitizing potential, because the latter is lacking. Accordingly, criteria for human data have been established that characterize 6 categories of human sensitizing potency, with 1 the most potent and 5 the least potent; category 6 represents true nonsensitizers. The literature has been searched, and 131 chemicals assigned into these categories according to their intrinsic potency judged only by the available human information. The criteria and data set generated provide a basis for examination of the capacity of nonanimal approaches for the determination of human sensitization potency.

Skin Sensitization to p-Phenylenediamine: The Diverging Roles of Oxidation and N-Acetylation for Dendritic Cell Activation and the Immune Response

Skin is a target of allergic reactions to aromatic amine hair dye precursors, such as p-phenylenediamine (PPD). As conversion of PPD on or in the skin is expected to be required for the induction of allergic contact dermatitis, we analyzed the role of oxidation and N-acetylation as major transformation steps. PPD and its oxidative and N-acetylated derivatives were tested for their sensitizing potential in vitro using a dendritic cell (DC) activation assay and in vivo using the local lymph node assay (LLNA). PPD did not induce relevant DC activation but induced a positive LLNA response. In contrast, DC activation was obtained when PPD was chemically pre-oxidized or after air oxygen exposure. Under both conditions, the potent sensitizing PPD oxidation product Bandrowskis base was identified along with other di- and trimeric species, indicating that PPD oxidation products provide an effective immune stimulation (danger signal). In contrast mono- and diacetylated PPD did not induce DC activation or a positive LLNA response. We conclude that dermal N-acetylation of PPD competes with the formation of oxidized PPD whereas skin exposure conditions allowing auto-oxidation, as in the LLNA, provide an effective danger signal necessary to induce skin sensitization to PPD.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: eye irritation.

Evaluation of the skin irritancy and corrosivity potential of an ingredient is a necessity in the safety assessment of cosmetic ingredients. To date, there are two formally validated alternatives to the rabbit Draize test for skin corrosivity in place, namely the rat skin transcutaneous electrical resistance (TER) assay and the Human Skin Model Test using EpiSkin, EpiDerm and SkinEthic reconstructed human epidermal equivalents. For skin irritation, EpiSkin, EpiDerm and SkinEthic are validated as stand-alone test replacements for the rabbit Draize test. Data from these tests are rarely considered in isolation and are evaluated in combination with other factors to establish the overall irritating or corrosive potential of an ingredient. In light of the deadlines established in the Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. In conclusion, the safety assessments for skin irritation/corrosion of new chemicals for use in cosmetics can be confidently accomplished using exclusively alternative methods.

Use of human in vitro skin models for accurate and ethical risk assessment: metabolic considerations.

Several human skin models employing primary cells and immortalized cell lines used as monocultures or combined to produce reconstituted 3D skin constructs have been developed. Furthermore, these models have been included in European genotoxicity and sensitization/irritation assay validation projects. In order to help interpret data, Cosmetics Europe (formerly COLIPA) facilitated research projects that measured a variety of defined phase I and II enzyme activities and created a complete proteomic profile of xenobiotic metabolizing enzymes (XMEs) in native human skin and compared them with data obtained from a number of in vitro models of human skin. Here, we have summarized our findings on the current knowledge of the metabolic capacity of native human skin and in vitro models and made an overall assessment of the metabolic capacity from gene expression, proteomic expression, and substrate metabolism data. The known low expression and function of phase I enzymes in native whole skin were reflected in the in vitro models. Some XMEs in whole skin were not detected in in vitro models and vice versa, and some major hepatic XMEs such as cytochrome P450-monooxygenases were absent or measured only at very low levels in the skin. Conversely, despite varying mRNA and protein levels of phase II enzymes, functional activity of glutathione S-transferases, N-acetyltransferase 1, and UDP-glucuronosyltransferases were all readily measurable in whole skin and in vitro skin models at activity levels similar to those measured in the liver. These projects have enabled a better understanding of the contribution of XMEs to toxicity endpoints.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. A COLIPA analysis.

For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies.

Comparative DNA microarray analysis of human monocyte derived dendritic cells and MUTZ-3 cells exposed to the moderate skin sensitizer cinnamaldehyde

The number of studies involved in the development of in vitro skin sensitization tests has increased since the adoption of the EU 7th amendment to the cosmetics directive proposing to ban animal testing for cosmetic ingredients by 2013. Several studies have recently demonstrated that sensitizers induce a relevant up-regulation of activation markers such as CD86, CD54, IL-8 or IL-1beta in human myeloid cell lines (e.g., U937, MUTZ-3, THP-1) or in human peripheral blood monocyte-derived dendritic cells (PBMDCs). The present study aimed at the identification of new dendritic cell activation markers in order to further improve the in vitro evaluation of the sensitizing potential of chemicals. We have compared the gene expression profiles of PBMDCs and the human cell line MUTZ-3 after a 24-h exposure to the moderate sensitizer cinnamaldehyde. A list of 80 genes modulated in both cell types was obtained and a set of candidate marker genes was selected for further analysis. Cells were exposed to selected sensitizers and non-sensitizers for 24 h and gene expression was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction. Results indicated that PIR, TRIM16 and two Nrf2-regulated genes, CES1 and NQO1, are modulated by most sensitizers. Up-regulation of these genes could also be observed in our recently published DC-activation test with U937 cells. Due to their role in DC activation, these new genes may help to further refine the in vitro approaches for the screening of the sensitizing properties of a chemical.

Introduction of a methoxymethyl side chain into p-phenylenediamine attenuates its sensitizing potency and reduces the risk of allergy induction

The strong sensitizing potencies of the most important primary intermediates of oxidative hair dyes, p-phenylenediamine (PPD) and p-toluylenediamine (PTD, i.e. 2-methyl-PPD) are well established. They are considered as the key sensitizers in hair dye allergic contact dermatitis. While modification of their molecular structure is expected to alter their sensitizing properties, it may also impair their color performance. With introduction of a methoxymethyl side chain we found the primary intermediate 2-methoxymethyl-p-phenylenediamine (ME-PPD) with excellent hair coloring performance but significantly reduced sensitizing properties compared to PPD and PTD: In vitro, ME-PPD showed an attenuated innate immune response when analyzed for its protein reactivity and dendritic cell activation potential. In vivo, the effective concentration of ME-PPD necessary to induce an immune response 3-fold above vehicle control (EC3 value) in the local lymph node assay (LLNA) was 4.3%, indicating a moderate skin sensitizing potency compared to values of 0.1 and 0.17% for PPD and PTD, respectively. Finally, assessing the skin sensitizing potency of ME-PPD under consumer hair dye usage conditions through a quantitative risk assessment (QRA) indicated an allergy induction risk negligible compared to PPD or PTD.

Cross Talk between Keratinocytes and Dendritic Cells: Impact on the Prediction of Sensitization

Understanding the mechanistic aspects involved in sensitization by chemicals will help to develop relevant preventive strategies. Many potential sensitizers are not directly immunogenic but require activation outside or inside the skin by nonenzymatic oxidation (prehaptens) or metabolic transformation (prohaptens) prior to being able to induce an immune response. This necessary activation step has not yet been actively integrated into a cell line-based prediction approach. We cocultured HaCaT keratinocytes with THP-1 as dendritic cell-like cells allowing intercellular interactions. The sensitizing potential was determined by analyzing differences in the expression of CD86, CD40, and CD54 on cocultured THP-1 cells. This new assay setup allowed (1) to distinguish irritants from allergens without influencing cell viability and (2) to discriminate pre/prohaptens from haptens. Under coculture conditions, the prohaptens eugenol, 2-methoxy-4-methylphenol, and benzo[a]pyrene induced a significantly higher upregulation of CD86 expression on THP-1. In agreement with the hapten concept, responses to 2,4-dinitrochlorobenzene, Bandrowskis base, and the prehapten isoeugenol were not significantly modified. Inhibition of cytochrome P450 or NAD(P)H:quinone oxidoreductase (NQO1) activity reduced the prohapten-mediated upregulation of CD86 on cocultured THP-1 cells. This coculture assay allowing cross talk between HaCaT and THP-1 cells appears to be suitable for the detection of prohaptens, is reproducible, easy to perform, and avoids donor variations. In addition, this assay is a promising approach to understand the impact of cross talk on the prediction of sensitization and once established may be integrated in a future in vitro toolbox to detect potential skin sensitizers and may thus contribute to reduce animal testing.

Extrahepatic metabolism at the body's internal–external interfaces

Abstract In general, xenobiotic metabolizing enzymes (XMEs) are expressed in lower levels in the extrahepatic tissues than in the liver, making the former less relevant for the clearance of xenobiotics. Local metabolism, however, may lead to tissue-specific adverse responses, e.g. organ toxicities, allergies or cancer. This review summarizes the knowledge on the expression of phase I and phase II XMEs and transporters in extrahepatic tissues at the bodys internal–external interfaces. In the lung, CYPs of families 1, 2, 3 and 4 and epoxide hydrolases are important phase I enzymes, while conjugation is less relevant. In skin, phase I-related enzymatic reactions are considered less relevant. Predominant skin XMEs are phase II enzymes, whereby glucuronosyltransferases (UGT) 1, glutathione-S-transferase (GST) and N-acetyltransferase (NAT) 1 are important for detoxification. The intestinal epithelium expresses many transporters and phase I XME with high levels of CYP3A4 and CYP3A5 and phase II metabolism is mainly related to UGT, NAT and Sulfotransferases (SULT). In the kidney, conjugation reactions and transporters play a major role for excretion processes. In the bladder, CYPs are relevant and among the phase II enzymes, NAT1 is involved in the activation of bladder carcinogens. Expression of XMEs is regulated by several mechanisms (nuclear receptors, epigenetic mechanisms, microRNAs). However, the understanding why XMEs are differently expressed in the various tissues is fragmentary. In contrast to the liver – where for most XMEs lower expression is demonstrated in early life – the XME ontogeny in the extrahepatic tissues remains to be investigated.