Application of the TTC concept to unknown substances found in analysis of foods

Unknown substances, not previously observed, are frequently detected in foods by quality control laboratories. In many cases, the assessment of these 'new' substances requires additional chemical analysis for their identification prior to assessing risk. This identification procedure can be time-consuming, expensive and in some instances difficult. Furthermore, in many cases, no toxicological information will be available for the substance. Therefore, there is a need to develop pragmatic tools for the assessment of the potential toxicity of substances with unknown identity to avoid delays in their risk assessment. Hence, the 'ILSI Europe expert group on the application of the threshold of toxicological concern (TTC) to unexpected peaks found in food' was established to explore whether the TTC concept may enable a more pragmatic risk assessment of unknown substances that were not previously detected in food. A step-wise approach is introduced that uses expert judgement on the source of the food, information on the analytical techniques, the dietary consumption of food sources containing the unknown substance and quantitative information of the unknown substance to assess the safety to the consumer using the TTC. By following this step-wise approach, it may be possible to apply a TTC threshold of 90. µg/day for an unknown substance in food. © 2011 Elsevier Ltd

Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.</p

Proteomic analysis of haptenation by skin sensitisers: Diphencyprone and ethyl acrylate

The potential risk of skin sensitisation, associated with the development of allergic contact dermatitis (ACD), is a consideration in the safety assessment of new ingredients for use in personal care products. Protein haptenation in skin by sensitising chemicals is the molecular initiating event causative of skin sensitisation. Current methods for monitoring skin sensitisation rely on limited reactivity assays, motivating interest in the development of proteomic approaches to characterise the skin haptenome. Increasing our mechanistic understanding of skin sensitisation and ACD using proteomics presents an opportunity to develop non-animal predictive methods and/or risk assessment approaches. Previously, we have used a novel stable isotope labelling approach combined with data independent mass spectrometry (HDMSE) to characterise the haptenome for a number of well-known sensitisers. We have now extended this work by characterising the haptenome of the sensitisers Diphenylcyclopropenone (DPCP) and Ethyl Acrylate (EA) with the model protein Human Serum Albumin (HSA) and the complex lysates of the skin keratinocyte, HaCaT cell line. We show that haptenation in complex nucleophilic models is not random, but a specific, low level and reproducible event. Proteomic analysis extends our understanding of sensitiser reactivity beyond simple reactivity assays and offers a route to monitoring haptenation in living cells

Determination of protein haptenation by chemical sensitisers within the complexity of the human skin proteome: protein-sensitiser haptenation of complex cellular lysates

Skin sensitisation associated with the development of allergic contact dermatitis (ACD) occurs via a number of specific key events at the cellular level. The molecular initiating event (MIE), the first in the sequence of these events, occurs after exposure of the skin to an electrophilic chemical, causing the irreversible haptenation of proteins within skin. Characterisation of this MIE is a key step in elucidating the skin sensitisation adverse outcome pathway and is essential to providing parameters for mathematical models to predict the capacity of a chemical to cause sensitisation. As a first step to addressing this challenge, we have exposed complex protein lysates from a keratinocyte cell line and human skin tissue with a range of well characterised sensitisers, including dinitrochlorobenzene (DNCB), 5-chloro-2-methylisothiazol-3-one (MCI), cinnamaldehyde (CA) and the non (or weak) sensitiser 6-methyl coumarin (6-MC). Using a novel stable isotope labelling approach combined with ion mobility assisted data independent mass spectrometry (HDMSE), we have characterised the haptenome for these sensitisers. Although a significant proportion of highly abundant proteins were haptenated, we also observed the haptenation of low abundant proteins by all three of the chemical sensitisers tested, indicating that within a complex protein background, protein abundance is not the sole determinant driving haptenation, highlighting a relationship to tertiary protein structure and the amino acid specificity of these chemical sensitisers and sensitiser potency. <br/