Margareta Törnqvist

Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman degradation method

Tissue doses of cancer initiators/mutagens are suitably monitored through hemoglobin adducts formed in vivo, but the use of this method has been hampered by a lack of sufficiently simple and fast procedures. It was previously observed that when the N-terminal amino acid in hemoglobin, valine, is alkylated it is cleaved off by the Edman sequencing reagent, phenyl isothiocyanate, in the neutral-alkaline coupling medium, as opposed to the acidic medium required by normal amino acids. Based on this principle, conditions for a functioning procedure for gas chromatography/mass spectrometry (GC/MS) determination of N-terminal alkylvalines in hemoglobin were worked out. Derivatizing the protein in formamide solution with pentafluorophenyl isothiocyanate, using a 2H-alkylated protein as internal standard, and applying on-column injection during analysis, permit reproducible determination of hydroxyethylvaline and other adducts down into the dose range where cancer risks may be considered acceptably low.

Investigations of factors that influence the acrylamide content of heated foodstuffs

The acrylamide content of heated foodstuffs should be considered to be the net result of complex reactions leading to the formation and elimination/degradation of this compound. The present study, involving primarily homogenized potato heated in an oven, was designed to characterize parameters that influence these reactions, including the heating temperature, duration of heating, pH, and concentrations of various components. Higher temperature (200 degrees C) combined with prolonged heating times produced reduced levels of acrylamide, due to elimination/degradation processes. At certain concentrations the presence of asparagine or monosaccharides (in particular, fructose and also glucose and glyceraldehyde) was found to increase the net content of acrylamide. Addition of other free amino acids or a protein-rich food component strongly reduced the acrylamide content, probably by promoting competing reactions and/or covalently binding acrylamide formed. The dependence on pH of the acrylamide content exhibited a maximum around pH 8; in particular, lower pH was shown to enhance elimination and decelerate formation of acrylamide. In contrast, the effects of additions of antioxidants or peroxides on acrylamide content were small or nonexistent.

Modified Edman degradation applied to hemoglobin for monitoring occupational exposure to alkylating agents

A method that can be used for the monitoring of exposure to alkylating agents in the environment is presented. It is based on the quantification of alkylated N‐terminal valines in hemoglobin, using a modified Edman degradation procedure. The detection limit (GC/electron capture detector or GC/MS) is increased by two magnitudes of ten when using pentafluorophenyl isothiocyanate instead of phenyl isothiocyanate in the derivatization step. As little as one nmole N‐(—2 hydroxyethyl)valine per gram hemoglobin can be detected under practical conditions.

Biological and chemical monitoring of occupational exposure to ethylene oxide.

Studies were carried out on two populations occupationally exposed to ethylene oxide (EtO) using different physical and biological parameters. Blood samples were collected from 9 hospital workers (EI) and 15 factory workers (EII) engaged in sterilization of medical equipment with EtO and from matched controls (CI and CII). Average exposure levels during 4 months (the lifespan of erythrocytes) prior to blood sampling were estimated from levels of N-(2-hydroxyethyl)valine adducts in hemoglobin. They were significantly enhanced in EI and EII and corresponded to a 40-h time-weighted average of 0.025 ppm in EI and 5 ppm in EII. Exposures were usually received in bursts with EtO concentrations in air ranging from 22 to 72 ppm in EI and 14 to 400 ppm in EII. All samples were analyzed for HPRT mutants (MFs), chromosomal aberrations (CAs), micronuclei (MN) and SCEs. MFs were significantly enhanced by 60% in EII but not in EI. These results are the first demonstration of mutation induction in man by ethylene oxide. CAs were significantly enhanced in EI and EII by 130% and 260% respectively. MN were not enhanced in EI but significantly in EII(217%). The mean frequency of SCEs was significantly elevated by 20% in EI and by almost 100% in EII. SCE was the only parameter that allowed distinction between daily and occasionally exposed workers in EII. An interesting finding in exposed workers was the large increase of the percentage of cells with high frequencies of SCE (3-4 times in EI and 17-fold in EII). The relative sensitivity of endpoints for detection of EtO exposure in the present investigation was in the following order: HOEtVal adducts greater than SCEs greater than chromosomal aberrations greater than micronuclei greater than HPRT mutants.

Induction of micronuclei in mouse and rat by glycidamide, genotoxic metabolite of acrylamide

Male CBA mice and male Sprague-Dawley rats were treated by i.p. injection of glycidamide (GA), the presumed genotoxic metabolite of acrylamide (AA). GA was obtained through a new way of synthesis. As an endpoint of chromosome damage, micronucleus (MN) induction in erythrocytes was measured. Hemoglobin (Hb) adducts were used as a measure of in vivo dose of GA. GA induced linear dose-dependent increases in adduct levels in both species. Rats exhibit, compared with mice, 30% higher Hb adduct levels per unit of administered amount of GA. The incremental MN frequencies per administered dose of GA in mice showed a linear-quadratic dose-dependent curve. In the rat no positive dose-response relationship was obtained, probably due to toxic effects to the bone marrow. The main result of this study is the finding that after treatment with synthetic GA the MN frequency per unit of the in vivo dose of GA in the mouse is very similar to that obtained in a previous study, where animals were treated with AA and GA as a metabolite. This equality in potency of GA, whether its in vivo dose is established by injection of synthetic GA or through metabolism of AA, supports the view that GA is the predominant genotoxic factor in AA exposure.

Degree of alkylation of macromolecules in vivo from variable exposure

Measurements of adducts formed with blood proteins, particularly haemoglobin, are increasingly being used to monitor human exposures to genotoxic chemicals. Information about the relationships between levels of genotoxic chemicals in the environment, e.g., concentration in the air, and levels of protein adducts in the blood is particularly important in setting safety standards and assessing risks. This paper describes the relationships between level of exposure to alkylating agents and level of haemoglobin adducts, considering the zero-order kinetics of the disappearance of these adducts. For comparison the corresponding relationship for adducts to macromolecules subjected to turnover, with first-order kinetics of disappearance, is described. For chemically stable and unstable adducts different exposure situations are considered: acute, chronic, intermittent and varying exposure levels. It is shown how an optimum solution of the problem of establishing the relationship between long-term exposure at varying levels (e.g., in work environments) and adduct level can be reached. Through mathematical derivations, which are given, expressions applicable to various exposure patterns are obtained and presented.

Acrylamide in food: mechanisms of formation and influencing factors during heating of foods

Background: In April 2002, the Swedish National Food Administration and a scientific group at the University of Stockholm jointly announced that they had shown acrylamide to be formed during the preparation of food and found it to occur in many foodstuffs. These new findings were clearly of concern to many types of industrial food processing as well as to home cooking. The Swedish Food Federation (Li) initiated and financed the formation of an expert committee to look into the chemical mechanisms. The present review is the final report of that expert committee. Design: The study identified, examined and put together facts and present knowledge on reaction routes for acrylamide formation in food and causal connections to cooking and food processing conditions. The results are based on literature surveys, examination of the analytical data published by the Swedish National Food Administration and other follow-up studies, contacts with international scientific networ ks, and observations from food companies. Results: The exact chemical mechanism(s) for acrylamide formation in heated foods is unknown. Several plausible mechanistic routes may be suggested, involving reactions of carbohydrates, proteins/amino acids, lipids and probably also other food components as precursors. With the data and knowledge available today it is not possible to point out any specific routes, or to exclude any possibilities. It is likely that a multitude of reaction mechanisms is involved. Acrolein is one strong precursor candidate, the origin of which could be lipids, carbohydrates or proteins/amino acids. Acrylamide is a reactive molecule and it can readily react with various other components in the food. The actual acrylamide level in a specific food product, therefore, probably reflects the balance between ease of formation and potential for further reactions in that food matrix. There are indications in support of that the Maillard reaction being an import a nt reaction route for acrylamide formation, but lipid degradation pathways to the formation of acrolein should also be considered. Conclusions: Reliable analytical methods to measure acrylamide in foods are available. Model studies are needed to identify precursors and reaction route(s) based on current hypotheses and to elucidate possible further reactions between acrylamide and other food components. Studies are needed to optimize formulation and processing conditions to minimize acrylamide levels, taking other product quality properties into consideration. Keywords: Acrylamide, cooking, food processing, heated foods, Maillard reaction.

Hemoglobin adducts and micronucleus frequencies in mouse and rat after acrylamide or N-methylolacrylamide treatment

The reactive industrial chemicals acrylamide (AA) and N-methylolacrylamide (MAA) are neurotoxic and carcinogenic in animals, MAA showing a lower potency than AA. The causative agent in AA-induced carcinogenesis is assumed to be the epoxy metabolite, glycidamide (GA), which in contrast to AA gives rise to stable adducts to DNA. The causative agent in MAA induced carcinogenesis is so far not studied. The two AAs were studied in mice and rats using analysis of hemoglobin (Hb) adducts as a measure of in vivo doses and the in vivo micronucleus (MN) assay as an end-point for chromosome damage. Male CBA mice were treated by intraperitoneal (i.p.) injection of three different doses and male Sprague-Dawley rats with one dose of each AA. Identical adducts were monitored from the two AAs [N-(2-carbamoylethyl)valine] and the respective epoxide metabolites [N-(2-carbamoyl-2-hydroxyethyl)valine]. Per unit of administered amount, AA gives rise to higher (three to six times) Hb adduct levels than MAA in mice and rats. Mice exhibit, compared with rats, higher in vivo doses of the epoxy metabolites, indicating that AAs were more efficiently metabolized in the mice. In mouse the two AAs induced dose-dependent increases in both Hb adduct level and MN frequency in peripheral erythrocytes. Per unit of administered dose MAA showed only half the potency for inducing micronuclei compared with AA, although the MN frequency per unit of in vivo dose of measured epoxy metabolite was three times higher for MAA than for AA. No increase in MN frequency was observed in rat bone marrow erythrocytes, after treatment with either AA. This is compatible with a lower sensitivity of the rat than of the mouse to the carcinogenic action of these compounds.

On cancer risk estimation of urban air pollution.

The usefulness of data from various sources for a cancer risk estimation of urban air pollution is discussed. Considering the irreversibility of initiations, a multiplicative model is preferred for solid tumors. As has been concluded for exposure to ionizing radiation, the multiplicative model, in comparison with the additive model, predicts a relatively larger number of cases at high ages, with enhanced underestimation of risks by short follow-up times in disease-epidemiological studies. For related reasons, the extrapolation of risk from animal tests on the basis of daily absorbed dose per kilogram body weight or per square meter surface area without considering differences in life span may lead to an underestimation, and agreements with epidemiologically determined values may be fortuitous. Considering these possibilities, the most likely lifetime risks of cancer death at the average exposure levels in Sweden were estimated for certain pollution fractions or indicator compounds in urban air. The risks amount to approximately 50 deaths per 100,000 for inhaled particulate organic material (POM), with a contribution from ingested POM about three times larger, and alkenes, and butadiene cause 20 deaths, respectively, per 100,000 individuals. Also, benzene and formaldehyde are expected to be associated with considerable risk increments. Comparative potency methods were applied for POM and alkenes. Due to incompleteness of the list of compounds considered and the uncertainties of the above estimates, the total risk calculation from urban air has not been attempted here.

Associations between estimated acrylamide intakes, and hemoglobin AA adducts in a sample from the Malmö Diet and Cancer cohort.

Objective:To examine the coherence of estimated intakes of acrylamide (AA) from foods, with hemoglobin (Hb) AA adduct levels, an objective marker of environmental AA exposure.Design:A cross-sectional study.Setting:The Malmö Diet and Cancer study, a large population-based prospective cohort (n=28 098) in the south of Sweden.Subjects:A sample of non-smoking (n=70) and smoking (n=72) women and men selected to obtain large variation in Hb AA adducts.Methods:Self-reported data on the usual consumption of foods were combined with published data on the AA content in Swedish foods. The Hb AA adduct levels were determined by a modified Edman degradation method. Linear regression and correlation analysis examined associations between estimated AA intakes, and Hb AA adducts.Results:In randomly selected individuals (n=40), the estimated median AA intake was 28 μg per day. In linear regression models, adjusting for sex, significant associations were seen in non-smokers between Hb AA adducts and estimated AA from foods (P=0.006). In smokers both AA from foods (P=0.006) and the calculated amount of tobacco consumed (P=0.003) were significantly associated with Hb AA adducts. Positive partial correlations between dietary AA estimates and Hb AA adducts were seen in smoking men (r=0.37) and women (r=0.59), and in non-smoking men (r=0.60), but not in non-smoking women.Conclusions:This study suggests that both diet and tobacco are important sources of the environmental AA exposure, although the lack of correlations in non-smoking women cast doubt on the validity of dietary AA intake estimates used in cancer epidemiology, or suggest that unrecognized factors may influence the internal dose measure of AA exposure.Sponsorship:Funded by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS); the European Commission (Contract no FOOD-CT-2003–506820, HEATOX); The Swedish Cancer Society; The Swedish Research Council; and The City of Malmö.