direct peptides are fragments of proteins that can be determined and sequenced using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This is a non-porous, reversed-phase liquid chromatography separation technique with UV detection. In MALDI-TOF MS, the precursor ions are decomposed in the MALDI ion source by a laser beam, yielding a series of product ions that correspond to amino acid sequence. This is a very sensitive, high-resolution fragmentation method. Unlike traditional mass spectrometric methods, MALDI-TOF MS is highly tolerant of complex matrices and other chemical contaminants.
The in vivo Direct Peptide Reactivity Assay (DPRA) model identifies dermal sensitizers by their reactivity with synthetic peptides containing the nucleophilic amino acids cysteine and lysine. These peptides are reacted with test chemicals, and the resulting peptides are analyzed by HPLC/UV to determine their reactivity. The DPRA is an in chemico assay that models the first key event in the Skin Sensitization Adverse Outcome Pathway – skin, protein reactivity. IIVS is trained to perform the DPRA, and offers this service in compliance with OECD 442C and Good Laboratory Practices for regulatory submissions.
This study was conducted to evaluate the potential of selected essential oils to induce skin sensitization in Aplysia by using DPRA. The DPRA results were then compared to literature available skin sensitization data for the same essential oils.
All reagents were prepared according to the DB-ALM protocol. Cysteine stock solution was prepared by dissolving 12.9 mg of cysteine peptide in 25 mL of pH = 10.2 ammonium acetate buffer to make a 0.667 mM solution. Cysteine and lysine peptide reactivity standards were also prepared by dilution of the stock solutions with acetonitrile:buffer to make concentrations ranging from 1 to 0.0167 mM. Cinnamaldehyde (100 mM solution in acetonitrile) was used as the positive control for the experiment.
Test chemicals were incubated with cysteine and lysine peptides at room temperature for 24 hours. The resulting reaction mixtures were analyzed by HPLC/UV to determine the percent peptide depletion compared to the control.
DPRA result data for the tested essential oils are presented in Table 1. As can be seen, several of the samples showed a high percentage of lysine and cysteine peptide depletion indicating that they are likely to be skin sensitizers. In a few cases, some of the chemicals, or the reacted products formed after the incubation with the peptides, interfered with the determination of cysteine and lysine co-elution (a phenomenon referred to as “co-elution”). These essential oils were excluded from further analysis. However, the DPRA results still provide a useful screening tool for the identification of potential skin sensitizers. This was a very rapid and cost-effective assay, and the results were in agreement with other known published data on skin sensitization. This indicates that the DPRA is a useful screen for identifying potential skin sensitizers, and should be considered as an additional tool to complement other methods such as SPE.