Andreas Natsch

Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro

In vitro tests are needed to replace animal tests to screen for the skin sensitization potential of chemicals. Skin sensitizers are electrophilic molecules and the Nrf2-electrophile-sensing pathway comprising the repressor protein Keap1, the transcription factor Nrf2 and the antioxidant response element (ARE) is emerging as a toxicity pathway induced by skin sensitizers. Previously, we screened a large set of chemicals in the reporter cell line AREc32, which contains an eight-fold repeat of the rat GSTA2 ARE-sequence upstream of a luciferase reporter gene in the human breast cancer cell line MCF7. This approach was now further developed to bring it closer to the conditions in the human skin and to propose a fully standardized assay. To this end, a luciferase reporter gene under control of a single copy of the ARE-element of the human AKR1C2 gene was stably inserted into HaCaT keratinocytes. A standard operating procedure was developed whereby chemicals are routinely tested at 12 concentrations in triplicate for significant induction of gene activity. We report results from this novel assay on (i) a list of reference chemicals published by ECVAM, (ii) the ICCVAM list of chemicals for validation of alternative endpoints in the LLNA and (iii) on a more general list of 67 chemicals derived from the ICCVAM database. For comparison, peptide reactivity data are presented for the same chemicals. The results indicate a good predictive value of this approach for hazard identification. Its technical simplicity, the high-throughput format and the good predictivity may make this assay a candidate for rapid validation to meet the tight deadline to replace animal tests for skin sensitization by 2013 set by the European authorities.

Biocontrol of soil-borne fungal plant diseases by 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads with different restriction profiles of amplified 16S rDNA

Fluorescent pseudomonads producing the antimicrobial compound 2,4-diacetylphloroglucinol (Phl) are being studied extensively for use as biocontrol agents of soil-borne fungal diseases. Some of them can produce pyoluteorin (Plt) in addition to Phl, whereas others synthesise only Phl. Here, a collection of seven Phl+ Plt- pseudomonads, seven Phl+ Plt+ pseudomonads and seven Phl- biocontrol pseudomonads were compared for protection of plant roots against fungal pathogens. The seven Phl+ Plt+ pseudomonads were identical by restriction analysis of amplified spacer ribosomal DNA (spacer ARDRA), whereas the Phl+ Plt- pseudomonads and especially the Phl- biocontrol pseudomonads were quite diverse by spacer ARDRA. Collectively, the Phl+ Plt- pseudomonads proved superior to the Phl+ Plt+ pseudomonads and the Phl- biocontrol pseudomonads for protection of tomato against Fusarium crown and root rot (in rockwool microcosms) or cucumber against Pythium damping-off (in non-sterile soil microcosms). There was no correlation between protection in vivo and inhibition of the corresponding fungal pathogen on plates. However, there was a significant correlation between the amount of Phl produced on plates and protection of tomato against Fusarium crown and root rot, but not with protection of cucumber against Pythium damping-off. Interestingly, the minority of strains unable to produce HCN, an extracellular protease, or both, were among those unable to protect plants in both pathosystems. A seedling assay was developed to compare pseudomonads for suppression of Fusarium crown and root rot in vitro, and a significant correlation was found between disease severity in vitro and in vivo. Overall, results suggest that promising biocontrol pseudomonads may be identified based on the ability to produce Phl and/or specific ARDRA-based fingerprints.

A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation.

Skin sensitization is a key endpoint for cosmetic ingredients, with a forthcoming ban for animal testing in Europe. Four alternative tests have so far been submitted to ECVAM prevalidation: (i) MUSST and (ii) h‐Clat assess surface markers on dendritic cell lines, (iii) the direct peptide reactivity assay (DPRA) measures reactivity with model peptides and (iv) the KeratinoSensTM assay which is based on detection of Nrf2‐induced luciferase. It is anticipated that only an integrated testing strategy (ITS) based on a battery of tests might give a full replacement providing also a sensitization potency assessment, but this concept should be tested with a data‐driven analysis. Here we report a database on 145 chemicals reporting the quantitative endpoints measured in a U937‐ test, the DPRA and KeratinoSensTM . It can serve to develop data‐driven ITS approaches as we show in a parallel paper and provides a view as to the current ability to predict with in vitro tests as we are entering 2013. It may also serve as reference database when benchmarking new molecules with in vitro based read‐across and find use as a reference database when evaluating new tests. The tests and combinations thereof were evaluated for predictivity, and overall a similar predictivity was found as before on three‐fold smaller datasets. Analysis of the dose–response parameters of the individual tests indicates a correlation to sensitization potency. Detailed analysis of chemicals false‐negative and false‐positive in two tests helped to define limitations in the tests but also in the database derived from animal studies. Copyright

LC-MS–Based Characterization of the Peptide Reactivity of Chemicals to Improve the In Vitro Prediction of the Skin Sensitization Potential

A key step in the skin sensitization process is the formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. Based on this mechanistic understanding, there is a renewed interest in in vitro assays to determine the reactivity of chemicals toward peptides in order to predict their sensitization potential. A standardized peptide reactivity assay yielded a promising predictivity. This published assay is based on high-performance liquid chromatography with ultraviolet detection to quantify peptide depletion after incubation with test chemicals. We had observed that peptide depletion may be due to either adduct formation or peptide oxidation. Here we report a modified assay based on both liquid chromatography-mass spectrometry (LC-MS) analysis and detection of free thiol groups. This approach allows simultaneous determination of (1) peptide depletion, (2) peptide oxidation (dimerization), (3) adduct formation, and (4) thiol reactivity and thus generates a more detailed characterization of the reactivity of a molecule. Highly reactive molecules are further discriminated with a kinetic measure. The assay was validated on 80 chemicals. Peptide depletion could accurately be quantified both with LC-MS detection and depletion of thiol groups. The majority of the moderate/strong/extreme sensitizers formed detectable peptide adducts, but many sensitizers were also able to catalyze peptide oxidation. Whereas adduct formation was only observed for sensitizers, this oxidation reaction was also observed for two nonsensitizing fragrance aldehydes, indicating that peptide depletion might not always be regarded as sufficient evidence for rating a chemical as a sensitizer. Thus, this modified assay gives a more informed view of the peptide reactivity of chemicals to better predict their sensitization potential.

Filling the Concept with Data: Integrating Data from Different In Vitro and In Silico Assays on Skin Sensitizers to Explore the Battery Approach for Animal-Free Skin Sensitization Testing

Tests for skin sensitization are required prior to the market launch of new cosmetic ingredients. Significant efforts are made to replace the current animal tests. It is widely recognized that this cannot be accomplished with a single in vitro test, but that rather the integration of results from different in vitro and in silico assays will be needed for the prediction of the skin sensitization potential of chemicals. This has been proposed as a theoretical scheme so far, but no attempts have been made to use experimental data to prove the validity of this concept. Here we thus try for the first time to fill this widely cited concept with data. To this aim, we integrate and report both novel and literature data on 116 chemicals of known skin sensitization potential on the following parameters: (1) peptide reactivity as a surrogate for protein binding, (2) induction of antioxidant/electrophile responsive element dependent luciferase activity as a cell-based assay; (3) Tissue Metabolism Simulator skin sensitization model in silico prediction; and (4) calculated octanol-water partition coefficient. The results of the in vitro assays were scaled into five classes from 0 to 4 to give an in vitro score and compared to the local lymph node assay (LLNA) data, which were also scaled from 0 to 4 (nonsensitizer/weak/moderate/strong/extreme). Different ways of evaluating these data have been assessed to rate the hazard of chemicals (Cooper statistics) and to also scale their potency. With the optimized model an overall accuracy for predicting sensitizers of 87.9% was obtained. There is a linear correlation between the LLNA score and the in vitro score. However, the correlation needs further improvement as there is still a relatively high variation in the in vitro score between chemicals belonging to the same sensitization potency class.

A Functional ABCC11 Allele Is Essential in the Biochemical Formation of Human Axillary Odor

The characteristic human axillary odor is formed by bacterial action on odor precursors that originate from apocrine sweat glands. Caucasians and Africans possess a strong axillary odor ,whereas many Asians have only a faint acidic odor. In this study, we provide evidence that the gene ABCC11 (MRP8), which encodes an apical efflux pump, is crucial for the formation of the characteristic axillary odor and that a single-nucleotide polymorphism (SNP) 538G --> A, which is prominent among Asian people, leads to a nearly complete loss of the typical odor components in axillary sweat. The secretion of amino-acid conjugates of human-specific odorants is abolished in homozygotic carriers of the SNP, and steroidal odorants and their putative precursors are significantly reduced. Moreover, we show that ABCC11 is expressed and localized in apocrine sweat glands. These data point to a key function of ABCC11 in the secretion of odorants and their precursors from apocrine sweat glands. SNP 538G --> A, which also determines human earwax type, is present on an extended haplotype, which has reached >95% frequency in certain populations in recent human evolution. A strong positive selection in mate choice for low-odorant partners with a dysfunctional ABCC11 gene seems a plausible explanation for this striking frequency of a loss-of-function allele.

Bayesian integrated testing strategy to assess skin sensitization potency: from theory to practice

Frameworks to predict in vivo effects by integration of in vitro, in silico and in chemico information using mechanistic insight are needed to meet the challenges of 21st century toxicology. Expert‐based approaches that qualitatively integrate multifaceted data are practiced under the term ’weight of evidence’, whereas quantitative approaches remain rare. To address this gap we previously developed a methodology to design an Integrated Testing Strategy (ITS) in the form of a Bayesian Network (BN). This study follows up on our proof of concept work and presents an updated ITS to assess skin sensitization potency expressed as local lymph node assay (LLNA) potency classes. Modifications to the ITS structure were introduced to include better mechanistic information. The parameters of the updated ITS were calculated from an extended data set of 124 chemicals. A detailed validation analysis and a case study were carried out to demonstrate the utility of the ITS for practical application. The improved BN ITS predicted correctly 95% and 86% of chemicals in a test set (n = 21) for hazard and LLNA potency classes, respectively. The practical value of using the BN ITS is far more than a prediction framework when all data are available. The BN ITS can develop a hypothesis using subsets of data as small as one data point and can be queried on the value of adding additional tests before testing is commenced. The ITS represents key steps of the skin sensitization process and a mechanistically interpretable testing strategy can be developed. These features are illustrated in the manuscript via practical examples. Copyright

High Throughput Kinetic Profiling Approach for Covalent Binding to Peptides: Application to Skin Sensitization Potency of Michael Acceptor Electrophiles

Research aimed at nonanimal approaches to provide the relevant information needed for the effective assessment of skin sensitization, for both hazard characterization and risk assessment purposes, is currently an area of high activity, stimulated by regulatory initiatives related to chemicals used in consumer products. The ability of a chemical to react covalently with protein or peptide nucleophiles in the skin is recognized as the key determinant in determining sensitization potency, and initiatives to develop peptide reactivity assays to replace animal testing have been undertaken recently. This paper describes a high throughput kinetic profiling (HTKP) approach, developed as an extension of a published standard assay, with the aim of providing a quantitatively robust end point in the form of a kinetic profile from which reactivity to a model peptide can be quantified in the form of second order rate constants. The approach allows solubility issues to be identified and overcome; these are frequently encountered, but can often go undetected, in aqueous reactivity assays with organic compounds of interest in the skin sensitization context. Using rate constants determined by the HTKP approach we have obtained a quantitative mechanistic model for the Michael acceptor reaction mechanistic domain, relating the sensitization potency in the murine local lymph node assay to the rate constant. The observation that the correlation is not improved by incorporation of a hydrophobicity term has implications regarding the nature and location of the skin nucleophile whose reaction leads to sensitization by Michael acceptor electrophiles.

Swiss Army Survey in Switzerland to Determine the Prevalence of Francisella tularensis, Members of the Ehrlichia phagocytophila Genogroup, Borrelia burgdorferi Sensu Lato, and Tick-Borne Encephalitis Virus in Ticks

Abstract A total of 6071 Ixodes ricinus ticks were collected on Swiss Army training grounds in five regions of Switzerland. The aim of the survey was to assess the prevalence of ticks infected with the human pathogens Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and the European tick-borne encephalitis virus. TaqMan PCR (PE Biosystems, USA) and TaqMan RT-PCR (PE Biosystems) analyses were performed on DNA and RNA extracted from pools of ten ticks grouped by gender. Here, for the first time, it is shown that ticks may harbor Francisella tularensis in Switzerland, at a rate of 0.12%. Furthermore, 26.54% of the ticks investigated harbored Borrelia burgdorferi sensu lato, 1.18% harbored members of the Ehrlichia phagocytophila genogroup, and 0.32% harbored the European tick-borne encephalitis virus. A new instrumentation was applied in this study to carry out and analyze more than 2300 PCR reactions in only 5 days. Furthermore, the results reveal that people working in outdoor areas, including army personnel on certain training grounds contaminated with ticks containing tick-borne pathogens, are at risk for different tick-borne diseases.

Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals.

The currently available animal-free methods for the detection of skin sensitizing potential of chemicals seem promising. However, no single method is able to comprehensively represent the complexity of the processes involved in skin sensitization. To ensure a mechanistic basis and cover the complexity, multiple methods should be integrated into a testing strategy, in accordance with the adverse outcome pathway that describes all key events in skin sensitization. Although current majority voting testing strategies have proven effective, the performance of individual methods is not taken into account. To that end, we designed a tiered strategy based on complementary characteristics of the included methods, and compared it to a majority voting approach. This tiered testing strategy was able to correctly identify all 41 chemicals tested. In terms of total number of experiments required, the tiered testing strategy requires less experiments compared to the majority voting approach. On the other hand, this tiered strategy is more complex due the number of different alternative methods required, and predicted costs are similar for both strategies. Both the tiered and majority voting strategies provide a mechanistic basis for skin sensitization testing, but the strategy most suitable for regulatory decision-making remains to be determined.