Birgit Paulsson

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.

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.

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.

Acrylamide exposure measured by food frequency questionnaire and hemoglobin adduct levels and prostate cancer risk in the Cancer of the Prostate in Sweden Study

Acrylamide, a probable human carcinogen, is formed during the cooking of many commonly consumed foods. Data are scant on whether dietary acrylamide represents an important cancer risk in humans. We studied the association between acrylamide and prostate cancer risk using 2 measures of acrylamide exposure: intake from a food frequency questionnaire (FFQ) and acrylamide adducts to hemoglobin. We also studied the correlation between these 2 exposure measures. We used data from the population‐based case‐control study Cancer of the Prostate in Sweden (CAPS). Dietary data was available for 1,499 cases and 1,118 controls. Hemoglobin adducts of acrylamide were measured in blood samples from a subset of 170 cases and 161 controls. We calculated odds ratios (ORs) for the risk of prostate cancer in high versus low quantiles of acrylamide exposure using logistic regression. The correlation between FFQ acrylamide intake and acrylamide adducts in non‐smokers was 0.25 (95% confidence interval: 0.14–0.35), adjusted for age, region, energy intake, and laboratory batch. Among controls the correlation was 0.35 (95% CI: 0.21–0.48); among cases it was 0.15 (95% CI: 0.00–0.30). The OR of prostate cancer for the highest versus lowest quartile of acrylamide adducts was 0.93 (95% CI: 0.47–1.85, p‐value for trend = 0.98). For FFQ acrylamide, the OR of prostate cancer for the highest versus lowest quintile was 0.97 (95% CI: 0.75–1.27, p trend = 0.67). No significant associations were found between acrylamide exposure and risk of prostate cancer by stage, grade, or PSA level. Acrylamide adducts to hemoglobin and FFQ‐measured acrylamide intake were moderately correlated. Neither measure of acrylamide exposure—hemoglobin adducts or FFQ—was associated with risk of prostate cancer.

Hemoglobin adducts as a measure of variations in exposure to acrylamide in food and comparison to questionnaire data

UNLABELLED Measurement of haemoglobin (Hb) adducts from acrylamide (AA) and its metabolite glycidamide (GA) is a possibility to improve the exposure assessment in epidemiological studies of AA intake from food. This study aims to clarify the reliability of Hb-adduct measurement from individual single samples for exposure assessment of dietary AA intake. The intra-individual variations of AA- and GA-adduct levels measured in blood samples collected over 20 months from 13 non-smokers were up to 2-fold and 4-fold, respectively. The corresponding interindividual variations observed between 68 non-smokers, with large differences in AA intake, were 6-fold and 8-fold, respectively. The intra-individual variation of the GA-to-AA-adduct level ratio was up to 3-fold, compared to 11-fold between individuals (n = 68). From AA-adduct levels the average AA daily intake (n = 68) was calculated and compared to that estimated from dietary history methodology: 0.52 and 0.67 μg/kg body weight and day, respectively. At an individual level the measures showed low association (Rs = 0.39). CONCLUSIONS Dietary AA is the dominating source to measured AA-adduct levels and corresponding inter- and intra-individual variations in non-smokers. Measurements from single individual samples are useful for calculation of average AA intake and its variation in a cohort, and for identification of individuals only from extreme intake groups.

Approach for cancer risk estimation of acrylamide in food on the basis of animal cancer tests and in vivo dosimetry.

The question about the contribution from acrylamide (AA) in food to the cancer risk in the general population has not yet had a satisfactory answer. One point of discussion is whether AA constitutes a cancer risk through its genotoxic metabolite, glycidamide (GA), or whether other mechanism(s) could be operating. Using a relative cancer risk model, an improvement of the cancer risk estimate for dietary AA can be obtained by estimation of the genotoxic contribution to the risk. One cornerstone in this model is the in vivo dose of the causative genotoxic agent. This paper presents an evaluation, according to this model, of published AA cancer tests on the basis of in vivo doses of GA in rats exposed in the cancer tests. The present status regarding data with importance for an improved estimation of the contribution from GA to the cancer risk of AA, such as in vivo doses measured in humans, is discussed.

Alcohol influence on acrylamide to glycidamide metabolism assessed with hemoglobin-adducts and questionnaire data

Our purpose was to investigate whether alcohol (ethanol) consumption could have an influence on the metabolism of acrylamide to glycidamide in humans exposed to acrylamide through food. We studied a subsample from a population-based case-control study of prostate cancer in Sweden (CAPS). Questionnaire data for alcohol intake estimates was compared to the ratio of hemoglobin-adduct levels for acrylamide and glycidamide, used as a measure of individual differences in metabolism. Data from 161 non-smoking men were processed with regard to the influence of alcohol on the metabolism of acrylamide to glycidamide. A negative, linear trend of glycidamide-adduct to acrylamide-adduct-level ratios with increasing alcohol intake was observed and the strongest association (p-value for trend=0.02) was obtained in the group of men with the lowest adduct levels (47 pmol/g globin) when alcohol intake was stratified by acrylamide-adduct levels. The observed trend is likely due to a competitive effect between ethanol and acrylamide as both are substrates for cytochrome P450 2E1. Our results, strongly indicating that ethanol influence metabolism of acrylamide to glycidamide, partly explain earlier observations of only low to moderate associations between questionnaire data on dietary acrylamide intake and hemoglobin-adduct levels.

In vitro studies of the influence of certain enzymes on the detoxification of acrylamide and glycidamide in blood.

Several enzymes involved in the metabolism of xenobiotic substances are polymorphic in humans. Inter-individual differences in response to certain chemicals, such as acrylamide, as a result of such genetic polymorphisms might affect health-risk assessments. Detoxification by, for example, conjugation with glutathione (GSH) will decrease the concentration. The dose of the compound and enzymes that enhance the conjugation with GSH will increase the detoxification rate. The dose of acrylamide or glycidamide has been measured in blood samples from individuals with defined genotypes for the glutathione transferases GSTT1 and GSTM1 after in vitro incubation with these compounds. The results indicate that these enzymes have no significant effect on the blood dose, measured as Hb adducts over time, after exposure to acrylamide or glycidamide.