Human axillary odor is known to be formed upon the action of Corynebacteria sp. on odorless axilla secretions. The known axilla odor determinant 3-methyl-2-hexenoic acid was identified in hydrolyzed axilla secretions along with a chemically related compound, 3-hydroxy-3-methylhexanoic acid. The natural precursors of both these acids were purified from non-hydrolyzed axilla secretions. From liquid chromatography/ mass spectrometry analysis, it appeared that the acids are covalently linked to a glutamine residue in fresh axilla secretions, and the corresponding conjugates were synthesized for confirmation. Bacterial isolates obtained from the human axilla and belonging to the Corynebacteria were found to release the acids from these odorless precursors in vitro. A Zn 2؉ -dependent aminoacylase mediating this cleavage was purified from Corynebacterium striatum Ax20, and the corresponding gene agaA was cloned and heterologously expressed in Escherichia coli. The enzyme is highly specific for the glutamine residue but has a low specificity for the acyl part of the substrate. agaA is closely related to many genes coding for enzymes involved in the cleavage of N-terminal acyl and aryl substituents from amino acids. This is the first report of the structure elucidation of precursors for human body odorants and the isolation of the bacterial enzyme involved in their cleavage.The axilla region of humans contains a dense arrangement of apocrine, eccrine, and sebaceous glands, and it is an everyday experience, that volatile substances emanating from these areas make a key contribution to human body odor. Although this odor is perceived by today's society as mainly unpleasant, several studies indicate that it may contain chemical signals that affect the menstrual cycle (1) or that may be involved in a major histocompatibility complex allele-dependent mate selection (2). These studies point to an important role of body odors in the evolutionary history of man.Sweat as it is secreted by axillary glands is odorless. Since the pioneering work of Shelley et al. (3), it is known that (a) the typical strong axilla odor can only be released from apocrine secretions, and (b) that the action of skin bacteria is needed to generate the odoriferous compounds from non-smelling molecules present in these secretions. Indeed, the axilla is a skin region supporting a dense bacterial population, which is dominated by the two genera Staphylococcus and Corynebacteria (4, 5). Most individuals carry a flora that is dominated by either one of these two genera, and a strong correlation was found between a high population of Corynebacteria and a strong axillary odor formation (4, 6). As a practical consequence of these findings, halogenated antibacterials and aluminum preparations for reducing the bacterial population have become the main active ingredient of commercial deodorants for the last 40 years. The scientific conclusion from this early work was that axilla secretions contain non-odoriferous precursors that must be transformed by bacterial en...

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.

Bioaccumulation in aquatic species is a critical end point in the regulatory assessment of chemicals. Few measured fish bioconcentration factors (BCFs) are available for fragrance ingredients. Thus, predictive models are often used to estimate their BCFs. Because biotransformation can reduce chemical accumulation in fish, models using QSAR-estimated biotransformation rates have been developed. Alternatively, biotransformation can be measured by in vitro methods. In this study, biotransformation rates for nine fragrance ingredients were measured using trout liver S9 fractions and used as inputs to a recently refined in vitro-in vivo extrapolation (IVIVE) model. BCFs predicted by the model were then compared to (i) in vivo BCFs, (ii) BCFs predicted using QSAR-derived biotransformation rates, (iii) BCFs predicted without biotransformation, and (iv) BCFs predicted by a well-known regression model. For fragrance ingredients with relatively low (<4.7) log K(OW) values, all models predicted BCFs below a bioaccumulation threshold of 1000. For chemicals with higher (4.7-5.8) log K(OW) values, the model incorporating measured in vitro biotransformation rates and assuming no correction for potential binding effects on hepatic clearance provided the most accurate predictions of measured BCFs. This study demonstrates the value of integrating measured biotransformation rates for prediction of chemical bioaccumulation in fish.

Three in vitro methods for the prediction of the skin sensitization hazard have been validated. However, predicting sensitizer potency is a key requirement for risk assessment. Here, we report a database of 312 chemicals tested in the KeratinoSens™ assay and for kinetic peptide binding. These data were used in multiple regression analysis against potency in the local lymph node assay (LLNA). The dataset covers the majority of chemicals from the validation of the LLNA to predict human potency and this subset was analyzed for prediction of human sensitization potency by in vitro data. Global analysis yields a regression of in vitro data to LLNA pEC3 with an R(2) of 60% predicting LLNA EC3 with a mean error of 3.5-fold. The highest weight in the regression has the reaction rate with peptides, followed by Nrf2-induction and cytotoxicity in KeratinoSens™. The correlation of chemicals tested positive in vitro with human data has an R(2) of 49%, which is similar to the correlation between LLNA and human data. Chemicals were then grouped into mechanistic domains based on experimentally observed peptide-adduct formation and predictions from the TIMES SS software. Predictions within these domains with a leave-one-out approach were more accurate, and for several mechanistic domains LLNA EC3 can be predicted with an error of 2- to 3-fold. However, prediction accuracy differs between domains and domain assignment cannot be made for all chemicals. Thus, this comprehensive analysis indicates that combining global and domain models to assess sensitizer potency may be a practical way forward.

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Axillary odor is known since 50 years to be formed upon the action of Corynebacteria on odorless axilla secretions, but the nature of the bacterial enzymes involved in this process remained a mystery. We identified the known axilla odor determinant 3-methyl-2-hexenoic acid in hydrolyzed axilla secretions along with a new, chemically related compound, 3-hydroxy-3-methyl-hexanoic acid. The natural, odorless precursors of both these acids were purified from non-hydrolyzed fresh axilla secretions. The malodorous acids were shown to be covalently linked to a glutamine residue in fresh axilla secretions. Corynebacteria, but not Staphylococci, isolated from the axilla were found to release the acids from these precursors in vitro. A Zn(2+) -dependent aminoacylase mediating this cleavage was then purified from Corynebacterium striatum Ax20 and the corresponding gene agaA was cloned and heterologously expressed in Escherichia coli. Based on these biochemical findings, novel approaches in research on axilla malodor control are presented: (a) With a new test method using the isolated Corynebacteria and their enzymatic activity, the direct malodor-controlling activity of existing cosmetic ingredients was evaluated. (b) The structure of the natural malodor precursor was modified by replacing the malodor acid with fragrance molecules. These new fragrance precursors were shown to be cleaved by the same aminoacylase.

Skin sensitizers chemically modify skin proteins rendering them immunogenic. Sensitizing chemicals have been divided into applicability domains according to their suspected reaction mechanism. The widely accepted Schiff base applicability domain covers aldehydes and ketones, and detailed structure-activity-modeling for this chemical group was presented. While Schiff base formation is the obvious reaction pathway for these chemicals, the in silico work was followed up by limited experimental work. It remains unclear whether hydrolytically labile Schiff bases can form sufficiently stable epitopes to trigger an immune response in the living organism with an excess of water being present. Here, we performed experimental studies on benzaldehydes of highly differing skin sensitization potential. Schiff base formation toward butylamine was evaluated in acetonitrile, and a detailed SAR study is presented. o-Hydroxybenzaldehydes such as salicylaldehyde and the oakmoss allergens atranol and chloratranol have a high propensity to form Schiff bases. The reactivity is highly reduced in p-hydroxy benzaldehydes such as the nonsensitizing vanillin with an intermediate reactivity for p-alkyl and p-methoxy-benzaldehydes. The work was followed up under more physiological conditions in the peptide reactivity assay with a lysine-containing heptapeptide. Under these conditions, Schiff base formation was only observable for the strong sensitizers atranol and chloratranol and for salicylaldehyde. Trapping experiments with NaBH₃CN showed that Schiff base formation occurred under these conditions also for some less sensitizing aldehydes, but the reaction is not favored in the absence of in situ reduction. Surprisingly, the Schiff bases of some weaker sensitizers apparently may react further to form stable peptide adducts. These were identified as the amides between the lysine residues and the corresponding acids. Adduct formation was paralleled by oxidative deamination of the parent peptide at the lysine residue to form the peptide aldehyde. Our results explain the high sensitization potential of the oakmoss allergens by stable Schiff base formation and at the same time indicate a novel pathway for stable peptide-adduct formation and peptide modifications by aldehydes. The results thus may lead to a better understanding of the Schiff base applicability domain.

The larvae of the specialist sawflyRhadinoceraea nodicornis Konow (Hymenoptera, Tenthredinidae) store in their hemolymph ceveratrum alkaloids originating from the host plantVeratrum album L. (Liliales, Melanthiaceae). The major alkaloid found in the hemolymph is 3-acetyl-zygadenine. Qualitative and quantitative data showed that the plant alkaloid 3-angeloylzygadenine is most probably metabolized in the larval gut to zygadenine and then acetylated. A still unidentified alkaloid with a molecular weight of 591 Da was detected in plant leaves as well as in the gut, hemolymph, and excrement of larvae. Protoveratrine A and B, on the other hand, seem to be degraded by the larvae. These findings indicate that the pathway of ceveratrum alkaloids inR. nodicornis larvae is fourfold: direct sequestration, metabolism followed by sequestration, excretion of intact alkaloids, and degradation. In contrast, no ceveratrum alkaloids were detected in the hemolymph and excrement of larvae of the generalist sawflyAglaostigma sp. fed withV. album leaves. Bioassays with the antMyrmica rubra L. proved that the hemolymph ofR. nodicornis larvae is highly deterrent and toxic. In bioassays evaluating defensive efficiency against predators (ants, spiders, and bushcrickets), no larvae were eaten. Ceveratrum alkaloids were also detected in the hibernating prepupae ofR. nodicornis. In feeding bioassays, the shrewCrocidura russula Hermann rarely fed upon prepupae, suggesting that this stage is also protected from predation to some degree. In field surveys, the only parasitoids recorded were two ichneumonid species that are believed to be specialized onR. nodicornis. Bioassays and field observations enable us to suppose thatR. nodicornis and its enemies produce a food web of ion connectance.