Method for measurement of exposure to electrophiles : Strategy for identification and exposure estimation using N-terminal haemoglobin adducts

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

Method for measurement of exposure to electrophiles : Strategy for identification and exposure estimation using N-terminal haemoglobin adducts

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

Internal Doses of Glycidol in Children and Estimation of Associated Cancer Risk

The general population is exposed to the genotoxic carcinogen glycidol via food containing refined edible oils where glycidol is present in the form of fatty acid esters. In this study, internal (in vivo) doses of glycidol were determined in a cohort of 50 children and in a reference group of 12 adults (non-smokers and smokers). The lifetime in vivo doses and intakes of glycidol were calculated from the levels of the hemoglobin (Hb) adduct N-(2,3-dihydroxypropyl)valine in blood samples from the subjects, demonstrating a fivefold variation between the children. The estimated mean intake (1.4 μg/kg/day) was about two times higher, compared to the estimated intake for children by the European Food Safety Authority. The data from adults indicate that the non-smoking and smoking subjects are exposed to about the same or higher levels compared to the children, respectively. The estimated lifetime cancer risk (200/105) was calculated by a multiplicative risk model from the lifetime in vivo doses of glycidol in the children, and exceeds what is considered to be an acceptable cancer risk. The results emphasize the importance to further clarify exposure to glycidol and other possible precursors that could give a contribution to the observed adduct levels

Red blood cell sedimentation rate measurements in a high aspect ratio microchannel

The erythrocyte sedimentation rate (ESR) test is commonly used in clinical practice for monitoring, screening and diagnosing pathological conditions and diseases related to the inflammatory response of the immune system. Several ESR techniques have been developed over the years improving the reliability, the precision and the duration of the measurement

Pathways to Identify Electrophiles In Vivo Using Hemoglobin Adducts : Hydroxypropanoic Acid Valine Adduct and Its Possible Precursors

Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level