Humans are daily exposed to chemicals from various sources, including cosmetics, jewelry, clothes, and hair dyes, which can result in the occurrence of contact allergy and subsequent allergic contact dermatitis (ACD), a type IV delayed hypersensitivity reaction. ACD is characterized by inflammation and eczema at the site of exposure, and no definitive cure for this condition has been identified to date, with only symptomatic treatment options involving corticosteroids being available.

The research presented in this thesis is centered around mass spectrometry (MS) strategies aimed at enhancing our comprehension of events that occur during the early stages of the development of contact allergy. Special emphasis is given to characterizing various contact allergens (haptens) and their interactions with endogenous proteins, as these interactions are considered crucial in the initiation of contact allergy. Moreover, the thesis endeavors to explore the activation of prehaptens and prohaptens, which are non-reactive compounds capable of transforming into haptens outside or inside the skin, respectively.

In Paper I, a bottom-up proteomics approach was employed to investigate the adductome of two major blood proteins, human serum albumin (HSA) and hemoglobin (Hb). The study aimed to identify the most reactive sites on these proteins upon exposure to different haptens with varying sensitization potencies. Highly susceptible sites on HSA and Hb were identified as the most likely targets for in vivo modification. This study is the first investigation of the Hb adductome in the context of contact allergy and may contribute to the development of improved diagnostic tools using blood samples. With Hb on focus, Paper II evaluated three different MS-based methods, including bottom-up proteomics, detachment of N-terminal adducts by FIRE, and limited proteolysis (LiP), to determine the most suitable approach for assessing exposure through this protein. The three methods showed different strengths and limitations depending on the nature of the hapten. In Paper III, the research conducted revealed the presence of a hapten-protein conjugate in blood samples mice treated with the synthetic hapten tetramethyl rhodamine isothiocyanate (TRITC) topically. The identified protein was the macrophage migration inhibitory factor (MIF), marking the first instance of such a conjugate being detected in blood samples after topical hapten application. The study also indicated that MIF could potentially be modified by other contact allergens, suggesting its potential as a biomarker for the condition. In Paper IV, contact allergy to propolis, a by-product of honey used in biocosmetics, was investigated. Air oxidation experiments with a model peptide and MS detection, revealed that quinones formed from the oxidation of major propolis components are responsible for adduct formation. The identified adducts are likely the cause of contact allergy to propolis, providing valuable insights into the underlying mechanisms of propolis contact allergy and potential implications for clinical diagnosis. In Paper V, the bioactivation of cinnamic alcohol, a common ingredient in many cosmetic products, was investigated using in vitro systems and a targeted MS approach. Two metabolites, namely pOH-cinnamic alcohol and pOH-cinnamic aldehyde, were identified as of particular interest and their sensitizing potency was evaluated, with the latter categorized as a moderate sensitizer.

In summary, this doctoral thesis employed MS techniques to characterize contact allergens and their protein conjugates, yielding valuable insights into the molecular mechanisms underlying contact allergy development. The findings have potential implications for improving diagnostic tools and strategies for preventing and treating contact allergy.

We are exposed to many chemicals in our everyday life. Some of these chemicals could pose risks for our health. To reduce such risks, it is important to know what we are exposed to, how much, and how toxic the chemicals are.

This thesis focuses on development and novel applications of a method for identifying and quantifying adducts as biomarkers of exposure to electrophiles. Electrophiles are reactive and can be measured as their stable reaction products (adducts) with haemoglobin (Hb) using the FIRE procedure™. This method utilizes Fluorescein Isothiocyanate to selectively cleave the adducted (R) N-terminal valine in human Hb, with a modified Edman procedure.

The primary aim was to further develop the FIRE procedure for application to a larger number of human blood samples, and to decrease the amount of sample needed. To achieve this, the method was adapted to 96-well plates. Further improvements resulted in a method that uses ca. half of the material used in the original method and can be used to analyse 1000 samples in one to two months.

The newly developed version of the FIRE procedure was applied to analyse blood from 144 Norwegian adults (Paper I) and 600 Swedish adolescents (Paper II), to estimate exposure to the electrophile acrylamide. IARC has classified acrylamide as a probable human carcinogen. Diet is a major source of exposure to acrylamide in the general population, as it is formed in carbohydrate-rich food during high temperature processing. Acrylamide intake was calculated from Hb adduct levels and compared with intake estimates calculated from self-reported food consumption data obtained from dietary records and food frequency questionnaires, combined with data for acrylamide content in food. In the Norwegian study, acrylamide intake was estimated by probabilistic calculations of the two types of food consumption data, which resulted in that no large difference in the median estimate obtained by these methods was observed in comparison of estimated daily intake from Hb adduct data (0.24-0.30 µg/kg body weight). In both studies (Papers I, II), the calculated margin of exposure with regard to risk for cancer indicates that acrylamide intake from food is of concern in the studied populations.

In Paper III, an unknown adduct, observed in earlier FIRE adductomics work, was identified with the help of high resolution accurate mass spectrometry, a synthesized standard, and nuclear magnetic resonance spectroscopy. A strategy to trace the source of the adduct was evaluated, and the epoxide glycidic acid was confirmed as a possible precursor, by measurement of adduct formation rate in vitro in human blood.

Finally, in Paper IV the FIRE procedure was compared to bottom-up proteomics to study Hb adducts from acrylamide, acrylic acid, glycidic acid, 2,3-epoxypropyl phenyl ether, 2-methyleneglutaronitrile, and 1-chloro-2,4-dinitrobenzene (DNCB). Adducts from all electrophiles were identified with bottom-up proteomics, with Cys93 in the beta chain of Hb as the most reactive side chain. The FIRE procedure was inefficient to detect bulkier/electron-withdrawing adducts, as from the contact allergen DNCB.

In conclusion, FIRE has high detectability for Hb adducts, although it is not suitable for adducts from all electrophiles. It is a valuable tool to identify and measure adducts, and to estimate exposure/intake of electrophiles. The new version of FIRE would facilitate the application for exposure measurement in epidemiological studies.