Cancer Risk Assessment of Glycidol : Evaluation of a Multiplicative Risk Model for Genotoxic Compounds

Humans are exposed to chemical compounds in everyday life, both from the environment and from endogenous processes. Some compounds constitute a risk for cancer development. One such compound is glycidol, which is genotoxic and an animal carcinogen. It is the model compound of this work, partly due to its presence in food. Glycidol, often together with 3-monochloropropane-1,2-diol (3-MCPD), occurs in the form of esters particularly in refined cooking oils, which are used in a variety of food products. The esters are hydrolyzed in the gastrointestinal tract to form glycidol (and 3-MCPD). The aim of the thesis has been to evaluate an approach for cancer risk estimation of genotoxic carcinogens based on a multiplicative (relative) risk model and genotoxic potency. Further, the aim was to estimate the cancer risk for exposure to glycidol via food. Measurement of the internal doses (concentration × time) of glycidol in the studied biological systems, including humans, has been crucial. Glycidol is electrophilic and forms adducts with nucleophilic sites in proteins and DNA. The doses of glycidol were quantified by mass spectrometry: in vivo from adduct levels to hemoglobin (Hb); in vitro from adducts to cob(I)alamin. The first part of the thesis concerns the genotoxic potency (genotoxic response per internal dose) of glycidol, measured in vitro by mutation studies and in vivo by micronuclei as a biomarker for genotoxicity (short-term studies in mice). The results were compared to that of ionizing radiation, used as a standard, to estimate the relative genotoxic potency of glycidol: 10 and 15 rad-equ./mMh from mutations and micronuclei, respectively. No induction of micronuclei was observed for the related compound 3-MCPD. Tumor incidence from published carcinogenicity studies of glycidol in mice and rats, together with the measured in vivo doses, was evaluated with the relative cancer risk model. A good agreement between predicted and observed tumor incidence was shown, and no significant difference of the obtained cancer risk coefficients (risk per dose) between mice (5.1 % per mMh) and rats (5.4 % per mMh) was observed. The overall results support that the relative risk coefficient (β) is independent of sex, tumor site, and species, and indicated that it can be transferred also to humans. The doubling dose, expressed as 1/β, is the dose that is required to double the background tumor incidence. The mean of the doubling doses from mice and rats (19 mMh) was assumed valid for risk estimation for humans. Transfer of β of glycidol to rad-equ. via its relative genotoxic potency showed a risk coefficient in agreement with the relative cancer risk coefficient of ionizing radiation. In the final work, the lifetime (70 years) in vivo doses of glycidol were calculated from measured Hb adduct levels in blood from 50 children and 12 adults, and compared to the doubling dose. A fivefold variation was observed in the in vivo doses. The estimated lifetime excess cancer risk from glycidol exceeds 1/1000. This is much higher than what is considered as an acceptable risk. To conclude, the multiplicative (relative) risk model together with relative genotoxic potency is promising to use in an approach for cancer risk estimation and in line with 3R (reduce-refine-replace) initiatives.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p

Rodenticide screening 2016–2018 Exposures in birds (raptors and gulls) and red foxes

Rodenticides are biocidal products that are used in order to control rats and mice. This screening study aims at investigating whether chemical substances belonging to the group anticoagulant rodenticides can be detected in Swedish non-target biota, and to investigate if the levels are different compared with the results from a previous study. The levels of anticoagulant rodenticides detected in the present screening study are similar to those found in earlier studies in Sweden and elsewhere. The literature indicates that toxic effects can occur in birds at levels &gt; 100 ng/g (liver) whereas the level &gt; 200 ng/g has been proposed to be a threshold level in foxes. Some individuals of raptors (n =2) and several foxes (n = 7) exceed these levels in the present study. These data suggest that anticoagulant rodenticides that are transferred in the food web may cause secondary toxicity in non-target mammals and birds in Sweden. However, no pathology has been performed for the individuals of the present study that can confirm any concentration-effect relationship or reason for mortality

Analysis of PFAS, phthalates, alternative plasticizers and organophosphate esters in sludge

Wastewater treatment plants (WWTPs) can be considered a water pollution point source as all potential pollutants from household as well as from certain industry production passes the WWTPs. Therefore, sludge from WWTPs may be enriched with pollutants and is a relevant matrix for screening for both known and hitherto unknown potential hazardous chemicals. If the sludge from municipal WWTPs is to be used as a source of nutrient or other purposes, then it could be necessary to employ methods for removal of micropollutants in the sludge. The present study constitutes an addition to a large-scale experiment focusing on the reduction of pharmaceuticals, antibiotics and hormones in sewage sludge stored over a period of a year. The objective of the present study was to analyse the presence of per- and polyfluoroalkyl substances (PFAS), organophosphate esters, phthalates and alternative plasticizers and their potential degradation/reduction in mesophilic anaerobic digested sludge in different treatments. This study is part of a larger on-going project on reduction of pharmaceuticals and organic pollutants in sludge. Extracts of sludge samples were analysed using three instrumental methods: liquid chromatography coupled to both tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS) and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Target analysis of several compounds (PFASs, organophosphate esters, phthalates and alternative plasticizers), analysis of total oxidizable precursors of PFASs and a suspect-screening of more than 1000 PFAS were performed at IVL. Extractable organic fluorine (EOF) was also performed on combustion ion chromatography at Stockholm University for quantification of potential unknown fluorinated compounds in the sludge. Decreasing trends for concentrations of organophosphate esters, phthalates, alternative plasticizers and PFAS could be observed in composted sludge over the storage time while the non-composted showed variable time trends for different substance classes. For PFAS, the sum concentrations of target analytes increased by almost an order of magnitude during 12 months of storage in the non-composted sludge. Furthermore, the results from TOP and EOF furthermore suggested that the sludge from both treatment experiments contained a significant fraction PFAS that could not be quantified by the targeted analysis.Avloppsreningsverk kan betraktas som en viktig punktkälla för föroreningar i vatten. Slam från avloppsreningsverk kan anrika föroreningar som har en huvudsaklig fördelning till slammet, och är således en relevant matris för screening för både kända och potentiella farliga kemikalier. Om slammet från kommunala avloppsreningsverk skall användas som en källa för näringsämnen eller andra syften kan det vara nödvändigt att avlägsna eller bryta ned potentiellt farliga mikroföroreningar i slammet. Denna studie utgör ett tillägg till ett storskaligt experiment som fokuserar på minskning av läkemedel, antibiotika och hormoner i avloppsslam som lagrats ett år. Kompakt eller poröst slam som rötats antingen termofilt eller mesofilt har i ovan nämnda experimentet lagrats under flera olika betingelser: täckt eller öppet, med och utan tillsats av urea, samt med och utan kompostering. I denna studie har endast poröst mesofilt rötat slam, som antingen har lagrats öppet eller med kompostering, studerats. Syftet med den här studien var att studera om per- och polyfluoralkylsubstanser (PFAS), organofosfatestrar samt ftalater och alternativa mjukgörare bryts ned under lagring. Extrakt av slamprover har analyserats med tre instrumentella metoder: vätskekromatografi kopplad till både tandemsmasspektrometri (LC-MS / MS) och högupplösta masspektrometri (LC-HRMS) samt gaskromatografi kopplad till tandemsmasspektrometri (GC-MS / MS). Förutom riktad kvantitativ analys av flera föreningar har analys av totala oxiderbara prekursorer av PFAS-ämnen och en sk suspect screening av mer än 1000 PFAS genomförts. Andelen extraherbart organiskt bunden fluor (EOF), dvs den totala fluormängden bundet till organiska ämnen, har också bestämts av Stockholms Universitet med förbränningsjonkromatografi för att kvantifiera andelen av potentiellt okända PFAS. Minskande trender för koncentrationer av organofosfatestrar, ftalater och alternativa mjukgörare samt PFAS kunde observeras i slam med kompostbehandlingen medan slammet som lagrats öppet inte visade någon tydlig trend för de ämnen som mättes i denna studie. För PFAS ökade summan av analyserade ämnen med nästan en tiopotens under 12 månaders lagring i det icke-komposterade slammet. Resultaten från TOP och EOF visade även på en betydande andel (91-97%) av hittills oidentifierade PFAS i slam från båda försöken

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 &#956;g/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

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

Applying a modified systematic review and integrated assessment framework (SYRINA) – a case study on triphenyl phosphate †

This work presents a case study in applying a systematic review framework (SYRINA) to the identification of chemicals as endocrine disruptors. The suitability and performance of the framework is tested with regard to the widely accepted World Health Organization definition of an endocrine disruptor (ED). The endocrine disrupting potential of triphenyl phosphate (TPP), a well-studied flame retardant reported to exhibit various endocrine related effects was assessed. We followed the 7 steps of the SYRINA framework, articulating the research objective via Populations, Exposures, Comparators, Outcomes (PECO) statements, performed literature search and screening, conducted study evaluation, performed data extraction and summarized and integrated the evidence. Overall, 66 studies, consisting of in vivo, in vitro and epidemiological data, were included. We concluded that triphenyl phosphate could be identified as an ED based on metabolic disruption and reproductive function. We found that the tools used in this case study and the optimizations performed on the framework were suitable to assess properties of EDs. A number of challenges and areas for methodological development in systematic appraisal of evidence relating to endocrine disrupting potential were identified; significant time and effort were needed for the analysis of in vitro mechanistic data in this case study, thus increasing the workload and time needed to perform the systematic review process. Further research and development of this framework with regards to grey literature (non-peer-reviewed literature) search, harmonization of study evaluation methods, more consistent evidence integration approaches and a pre-defined method to assess links between adverse effect and endocrine activity are recommended. It would also be advantageous to conduct more case studies for a chemical with less data than TPP

Adductomic Screening of Hemoglobin Adducts and Monitoring of Micronuclei in School-Age Children

Electrophilic compounds/metabolites present in humans, originating from endogenous processes or exogenous exposure, pose a risk to health effects through their reactions with nucleophilic sites in proteins and DNA, forming adducts. Adductomic approaches are developed to screen for adducts to biomacromolecules in vivo by mass spectrometry (MS), with the aim to detect adducts corresponding to unknown exposures from electrophiles. In the present study, adductomic screening was performed using blood samples from healthy children about 12 years old (<i>n</i> = 51). The frequencies of micronuclei (MN) in erythrocytes in peripheral blood were monitored as a measure of genotoxic effect/genotoxic exposure. The applied adductomic approach has been reported earlier by us and is based on analysis of N-terminal valine adducts in hemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC-MS/MS). High resolution MS was introduced for refined screening of previously unknown N-terminal Hb adducts. Measured adduct levels were compared with MN frequencies using multivariate data analysis. In the 51 individuals, a total of 24 adducts (whereof 12 were previously identified) were observed and their levels quantified. Relatively large interindividual variations in adduct levels were observed. The data analysis (with partial least-squares regression) showed that as much as 60% of the MN variation could be explained by the adduct levels. This study, for the first time, applies the combination of these sensitive methods to measure the internal dose of potentially genotoxic chemicals and genotoxic effects, respectively. The results indicate that this is a valuable approach for the characterization of exposure to chemical risk factors for the genotoxic effects present in individuals of the general population