Genotoxic Metabolite Glycidamide Research Articles

Abstract 1477: Acrylamide exposure in obesity enhances mammary epithelial DNA damage via the activity of its metabolite, glycidamide, and increased oxidative stress

Abstract Obesity is a world-wide epidemic, with 39% of adults considered overweight and 13% obese, and rates are rising. Obese women are at greater risk for developing breast cancer than lean women, however the mechanisms of how obesity increases breast cancer risk are not well understood. There is also limited knowledge on how environmental toxins may interact with obesity to promote breast cancer. Acrylamide, a probable carcinogen, is a biproduct in starchy foods cooked at high temperatures, which are prevalent in the obesity-inducing Western diet. Acrylamide is metabolized by the CYP2E1 enzyme into the epoxide, glycidamide, which is known to induce DNA damage. CYP2E1 activity is elevated in obesity, which could enhance DNA damage in obese individuals. Acrylamide exposure is associated with an increased risk of breast cancer in epidemiologic studies, but the evidence has been contradictory. To investigate how acrylamide impacts DNA damage in the obese mammary gland, three-week-old FVB female mice were randomized to receive a low-fat (LFD; 16% kcal from fat) or high-fat diet (HFD; 60% kcal from fat) and either 0.7 mM acrylamide water or control water. HFD-fed mice gained significantly more weight than LFD-fed mice, and exposure to acrylamide did not impact weight gain compared to respective controls. Mammary epithelial cells from acrylamide-treated mice had increased DNA damage compared to controls measured by COMET assays, with the highest DNA damage present in epithelial cells from obese acrylamide-treated mice. Mammary epithelial cells from acrylamide-treated mice also had enhanced oxidative DNA damage, measured by 8-OHdG adducts, and an additive effect was observed between acrylamide exposure and obesity. To investigate the impact of acrylamide in vitro, COMMA-D mammary epithelial cells were treated with vehicle, 9.8 µM acrylamide, or 0.5 mM glycidamide for 24 hours, then COMET assays and immunohistochemistry for markers of DNA damage were performed. COMMA-D cells treated with acrylamide-metabolite glycidamide showed significantly increased double strand DNA breaks compared to vehicle-treated cells, however acrylamide-treated cells did not. Interestingly, acrylamide-treated cells demonstrated significantly higher levels of intracellular reactive oxygen species compared to both glycidamide and vehicle-treated cells. These studies suggest acrylamide exposure enhances DNA damage in mammary epithelial cells via conversion to the genotoxic metabolite glycidamide and through generation of oxidative stress, which is exacerbated by obese conditions. These studies suggest that long-term acrylamide exposure through foods common in the Western diet may enhance DNA damage in mammary epithelial cells, potentially enhancing obesity-induced breast cancer risk. Citation Format: Brenna Walton, Lisa Arendt. Acrylamide exposure in obesity enhances mammary epithelial DNA damage via the activity of its metabolite, glycidamide, and increased oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1477.

Transcriptional profiling of male CD-1 mouse lungs and Harderian glands supports the involvement of calcium signaling in acrylamide-induced tumors

Acrylamide (AA) exposure causes increased incidence of forestomach, lung, and Harderian gland tumors in male mice. One hypothesized mode of action (MOA) for AA-carcinogenicity includes genotoxicity/mutagenicity as a key event, possibly resulting from AA metabolism to the direct genotoxic metabolite glycidamide. Alternatively, altered calcium signaling (CS) has been proposed as a central key event in the MOA. To examine the plausibility of these proposed MOAs, RNA-sequencing was performed on tumor target tissues: Harderian glands (the most sensitive tumor target tissue in the rodent 2-year cancer bioassay) and lungs of AA-exposed male CD-1 mice. Animals were exposed to 0.0, 1.5, 3.0, 6.0, 12.0, or 24.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. We observed a pronounced effect on genes involved in CS and cytoskeletal processes in both tissues, but no evidence supporting a genotoxic MOA. Benchmark dose modeling suggests transcriptional points of departure (PODs) of 0.54 and 2.21 mg/kg bw-day for the Harderian glands and lungs, respectively. These are concordant with PODs of 0.17 and 1.27 mg/kg bw-day derived from the cancer bioassay data for these tissues in male mice, respectively. Overall, this study supports the involvement of CS in AA-induced mouse carcinogenicity, which is consistent with a recently proposed CS-based MOA in rat thyroid, and with other published reports of aberrant CS in malignant tumors in rodents and humans.

Differences in micronucleus frequency and acrylamide adduct levels with hemoglobin between vegetarians and non-vegetarians.

Nutrients and food constituents can prevent or contribute to genotoxicity. In this study, the possible influence of a vegetarian/non-vegetarian diet on genotoxic effects was investigated in 58 non-smoking healthy vegetarians (V) and non-vegetarians (NV), age 21-37 years from the Stockholm area in Sweden. Physical activity and dietary habits were similar in both groups, with the exception of the intake of meat and fish. Using flow cytometry, we determined the formation of micronuclei (MN) in transferrin-positive immature peripheral blood reticulocytes (Trf-Ret) (Total: n = 53; V: n = 27; NV: n = 26). Dietary exposure to acrylamide was measured through hemoglobin (Hb) adducts in peripheral erythrocytes (Total: n = 53; V: n = 29; NV: n = 24). Hb adducts of both acrylamide and its genotoxic metabolite glycidamide were monitored as a measure of the corresponding in vivo doses. Our data demonstrated that compared with the non-vegetarians, the vegetarians exhibited lower frequencies of MN (fMN) in the Trf-Ret (p < 0.01, Student's t test). A multivariate analysis demonstrated that there was no association between the fMN and factors such as age, sex, intake of vitamins/minerals, serum folic acid and vitamin B12 levels, physical activity, and body mass index. The mean Hb adduct levels of acrylamide and glycidamide showed no significant differences between vegetarians and non-vegetarians. Furthermore, there were no significant relationships between the adduct levels and fMN in the individuals. The ratio of the Hb adduct levels from glycidamide and acrylamide, however, showed a significant difference (p < 0.04) between the two groups. These data suggest that the vegetarian diet might be beneficial in lowering genomic instability in healthy individuals. The measured Hb adduct levels indicate that the total intake of acrylamide does not differ between the two studied groups and does not contribute to the observed difference in fMN, although an influence of the diet on the metabolic rates of acrylamide was indicated. In addition, the observed significant difference in the background fMN in the two groups demonstrated that the MN analysis method has a sensitivity applicable to the biomonitoring of human lifestyle factors.

Abstract 5732: Mercapturic acid excretion and N7-GA-Gua DNA adduct formation after low dose oral application of acrylamide to rats

Abstract Acrylamide (AA), a genotoxic carcinogen (IARC class 2A), is a food contaminant formed by thermal treatment of food. The margin of exposure (MOE) of AA, representing the distance between the bench mark dose associated with 10% tumor incidence in rats and the estimated average human exposure, is considered to be low and of human health concern. After uptake, AA is partly converted by CYP450 2E1 into the genotoxic epoxide glycidamide (GA). GA forms DNA adducts, primarily at N7 of guanine (N7-GA-Gua). AA and its genotoxic metabolite GA are conjugated to glutathione and excreted via urine as mercapturic acids (MA), namely N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA) which are used as biomarkers for exposure. Female Sprague Dawley (SD) rats were kept on an AA free diet. AA was given by gavage in a broad dosage range, covering single doses of 0.1-10,000 µg/kg bw. Formation of urinary MAs and of N7-GA-Gua DNA adducts in liver, kidney and lung was measured 16 h after application. The lowest dosage of 0.1 µg AA/kg bw did not result in significantly enhanced MA excretion, as compared to untreated controls, found to excrete about 0.8 nmol MA. Since the animals may have ingested at best 0.4 nmol AA/d with the diet, this findings suggests a background level of endogenously formed AA. At the lowest dosage (0.1 µg AA/kg bw), N7-GA-Gua adducts above the limit of detection (LOD, 0.2 adducts/10exp8 nucleotides) were not detected in any organ tested. At 1 µg/kg bw, enhanced adduct levels were found in kidney (around 1 adduct/10exp8 nucleotides) and lung (&amp;lt; 1 adduct/10exp8 nucleotides), but not in liver. At 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg AA/kg bw (about 1-2 adducts/10exp8 nucleotides). Taken together, the results of this in vivo study allow to conclude that exposure to single doses of AA in the range of human dietary exposure leads to N7-GA-Gua adduct levels in tissues of SD rats at the low end of steady state background DNA lesions of various origin in human and rat tissues. This study was supported by ISIC, the Institute for Scientific Information on Coffee. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5732. doi:1538-7445.AM2012-5732

In Vivo Doses of Acrylamide and Glycidamide in Humans after Intake of Acrylamide-Rich Food

For assessment of cancer risk from acrylamide (AA) exposure through food, the relation between intake from food in humans and the in vivo doses (area under the concentration-time curve, AUC) of AA (AUC-AA) and of its genotoxic metabolite glycidamide (GA) (AUC-GA) is used as a basis for extrapolation between exposure levels and between species. In this study, AA-rich foods were given to nonsmokers: a high intake of 11 μg AA/kg body weight (bw) and day for 4 days or an extra (medium) intake of 2.5 μg AA/kg bw and day for a month. Hemoglobin (Hb)-adduct levels from AA and GA, measured in blood samples donated before and after exposures, were used for calculation of AUC-AA and AUC-GA using reaction rate constants for the adduct formation measured in vitro. Both AA- and GA-adduct levels increased about twofold after the periods with enhanced intake. AUC for the high and medium groups, respectively, in nanomolar hours per microgram AA per kilogram bw, was for AA 212 and 120 and for GA 49 and 21. The AA intake in the high group was better controlled and used for comparisons with other data. The AUCs per exposure dose obtained in the present human study (high group) are in agreement with those previously obtained at 10(2) times higher exposure levels in humans. Furthermore, the values of AUC-AA and AUC-GA are five and two times higher, respectively, than the corresponding values for F344 rats exposed to AA at levels as in published cancer bioassays.

Simultaneous determination of acrylamide, its metabolite glycidamide and antipyrine in human placental perfusion fluid and placental tissue by liquid chromatography–electrospray tandem mass spectrometry

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of acrylamide (AA) and its genotoxic metabolite glycidamide (GA) with a test marker antipyrine (AP) in placental tissue and perfusion medium used in human placental perfusion studies. An internal standard ( 13C-acrylamide) was added to the samples which were then deproteinized with acetonitrile. Chromatographic separation was performed on a reversed phase column with a gradient elution of acetonitrile and 0.01% formic acid at a flow rate of 0.3 mL/min. Detection and quantification of the analytes were carried out with a triple quadrupole mass spectrometer using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The method was validated and linear over a concentration range of 0.5–20 μg/mL for acrylamide and glycidamide and 5–200 μg/mL for antipyrine. The lower limit of quantification for acrylamide and glycidamide was 0.5 μg/mL and for antipyrine 5 μg/mL. The method was selective, and good accuracy, precision, recovery, and stability were obtained for concentrations within the standard curve. The method was successfully used to analyze the placental perfusion medium and tissue samples in a toxicokinetic study for transplacental transfer of acrylamide and glycidamide. This is the first time that acrylamide, glycidamide and antipyrine are measured simultaneously.

Transplacental transfer of acrylamide and glycidamide are comparable to that of antipyrine in perfused human placenta

Most drugs can penetrate the placenta but there are only a few studies on placental transfer of environmental toxic compounds. In this study, we used dual recirculating human placental perfusion to determine the transfer rate through the placenta of a neurotoxic and carcinogenic compound found in food, acrylamide and its genotoxic metabolite glycidamide. Putative acrylamide metabolism into glycidamide during the 4-h perfusions and acrylamide-derived DNA adducts in placental DNA after perfusions were also analyzed. Placentas were collected immediately after delivery and kept physiologically functional as confirmed by antipyrine kinetics, glucose consumption and leak from fetal to maternal circulation. Acrylamide (5 or 10 μg/ml) or glycidamide (5 μg/ml), both with antipyrine (100 μg/ml), was added to maternal circulation. Acrylamide and glycidamide were analyzed in the perfusion medium by liquid chromatography/mass spectrometry. Acrylamide and glycidamide crossed the placenta from maternal to fetal circulation with similar kinetics to antipyrine, suggesting fetal exposure if the mother is exposed. The concentrations in maternal and fetal circulations equilibrated within 2 h for both studied compounds and with both concentrations. Acrylamide metabolism into glycidamide was not detected during the 4-h perfusions. Moreover, DNA adducts were undetectable in the placentas after perfusions. However, fetuses may be exposed to glycidamide after maternal metabolism. Although not found in placental tissue after 4 h of perfusion, it is possible that glycidamide adducts are formed in fetal DNA.

Internal doses of acrylamide and glycidamide in mice fed diets with low acrylamide contents

The formation of acrylamide during heating of certain foodstuffs constitutes a potential health hazard. The health risk assessment should be based on knowledge about the relation between dietary exposure to acrylamide and internal doses of acrylamide and its genotoxic metabolite glycidamide. The primary aim of this study in mice was to measure these relationships at low levels of acrylamide intake through the diet. A secondary aim was to clarify which extraction method should be used when analyzing acrylamide in food in order to obtain a correct measure of the acrylamide that is available for absorption. In the analysis procedure, alkaline extraction has earlier shown much higher measured acrylamide levels in certain foods compared to water extraction. In this subcronic study the administered diets were composed to give five levels of acrylamide intakes between 3 and 50 mug/kg body weight per day (calculated on figures obtained after water extraction). Internal doses of acrylamide and glycidamide were measured through hemoglobin (Hb)-adducts. The results showed linear relationships between the exposure of acrylamide and Hb-adduct levels from both acrylamide and glycidamide at these low exposure levels. The study also showed that the "extra" acrylamide measured with alkaline extraction does not correspond to bioavailable acrylamide.

Genotoxicity of Glycidamide in Comparison to 3-N-Nitroso-oxazolidin-2-one

Acrylamide (AA) is generated by thermal processing of foods, depending on processing conditions and precursor availability. AA is not genotoxic by itself but becomes activated to its genotoxic metabolite glycidamide (GA) via epoxidation, mediated primarily by cytochrome P450 2E1. In the Comet assay in V79 cells and in human lymphocytes, GA induced DNA damage down to 300 microM concentration (4 h). After post-treatment with the DNA repair enzyme formamidopyrimidine-DNA-glycosylase (FPG), DNA damage became already detectable at 10 microM (4 h). By comparison, the N-nitroso compound 3- N-nitroso-oxazolidin-2-one (NOZ-2) is a much stronger genotoxic agent, significantly inducing DNA damage already at 15 min (3 microM). Post-treatment with FPG in this case did not enhance response. GA induced DNA damage in V79 cells rather slowly, with first response detectable at 4 h. The hPRT forward mutation test encompasses 5 days of expression time during which also repair can take place. GA-induced hPRT mutations only became detectable at concentrations of 800 microM and above. This is 80-fold higher than the lowest significant response to GA in the Comet assay (10 microM with FPG). In contrast, NOZ-2 was as effective in the hPRT test as in the Comet assay (3 microM). These results demonstrate substantial differences in the genotoxic potency of GA and NOZ-2. Whereas NOZ-2 is a pontent genotoxic mutagen, GA in comparison shows only low genotoxic and mutagenic potential, presumably as a result, at least in part, of preferential N7-G alkylation.

Acrylamide in children – exposure assessment via urinary acrylamide metabolites as biomarkers

Acrylamide (AA), a substance classified as probably carcinogenic to humans, was detected for the first time in food products in 2002. AA can be primarily found in foods containing carbohydrates and proteins, where it is formed during the heating process. Exposure assessment based on food consumption data revealed an average daily intake of AA between 0.3 and 0.8 microg/kg BW/day. These data have been confirmed by human biomonitoring using haemoglobin adducts of AA in blood or the specific mercapturic acids in urine. However, human biomonitoring data on the internal exposure of children were only sporadically available. Especially data about the excretion of both relevant mercapturic acids were missing. The mercapturic acids other than the haemoglobin adducts give the recent AA exposure of the last 24h. In this study, we quantify the internal exposure of AA and the genotoxic metabolite glycidamide (GA) in 110 children with regard to their exposure through diet and/or environmental tobacco smoke. Hundred and ten 5-6-year-old children were randomly selected. Their dietary habits as well as their exposure to the environmental tobacco smoke were assessed by means of a questionnaire. By means of spot urine samples, mercapturic acids of acrylamide (AAMA) and mercapturic acids of glycidamide (GAMA) were analysed with LC-ESI-MS/MS. Median (95th percentile) urinary levels were 36.0 (152.7) microg AAMA/l and 13.4 (55.9) microg GAMA/l. Based on the metabolite levels, the median uptake of acrylamide was calculated to be 0.54 microg/kg BW/d. A number of associations with the consumption of French fries, various potato products, as well as fried cereals could be found. Significant results were found for French fries. No correlations between the exposure to environmental smoke and cotinine levels in urine were found. This is the first study to show the presence of AAMA and GAMA in urine specimens of 110 children, thus providing evidence for a background exposure by nutrition. Median (95th percentile) uptake of AA in children was 0.54 (1.91) microg/kg bodyweight and day, exceeding exposure in adults by 50%. These findings support the efforts to minimize AA formation and contamination in food. Comparing our findings with that of other human studies, there are hints that children have a higher AA intake than adults and that children more effectively oxidize AA. Both findings indicate that children might be the most vulnerable group of the population.

Comparison of Estimated Dietary Intake of Acrylamide with Hemoglobin Adducts of Acrylamide and Glycidamide

In a study comprising 50 subjects, we investigated the relationship between acrylamide (AA) intake from food using food frequency questionnaires and the concentration of hemoglobin (Hb) adducts of AA and its genotoxic metabolite glycidamide (GA) as a measure of the internal exposure. A method using solid-phase extraction and liquid chromatography with negative electrospray tandem mass spectrometric (MS/MS) detection for the determination of the Hb adducts as phenylthiohydantoin derivatives in human blood was developed. The limit of quantification for AA- and GA-Hb adducts were 2 and 6 pmol/g globin, respectively, and the between-assay precision was below 25%. The estimated dietary intake of AA was (median and range) 13.5 microg/day (4.1-72.6) in nonsmokers and 18.3 microg/day (7.8-32.0) in smokers. In nonsmokers, males had a higher intake than females, 16.6 microg/day (18.6-72.6) and 12.8 microg/day (4.1-30.2), respectively. Nonsmokers had a median AA and GA adduct concentration of 36.8 (range 17.9-65.5) and 18.2 (range 6.7-45.6) pmol/g globin, respectively. In smokers, the values were 165.8 (98.8-211) and 83.2 (29.1-99.0) pmol/g globin, respectively. Using multiple linear regression analysis, a significant positive correlation was found between the AA-Hb adduct concentration and the intake of chips/snacks and crisp bread. GA-Hb adduct did not correlate with consumption of any of the main food groups. Neither AA-Hb nor GA-Hb adduct concentration correlated with total dietary intake of AA as calculated from the reported food intake. Adduct concentrations did not correlate with 24 h urinary excretion of mercapturic acid metabolites of AA and GA in the same subjects reported previously.

Excretion of mercapturic acids of acrylamide and glycidamide in human urine after single oral administration of deuterium-labelled acrylamide

We investigated the human metabolism of AA to the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L: -cysteine (GAMA) which are derived from AA itself and from its oxidative genotoxic metabolite glycidamide (GA), respectively. A healthy male volunteer received a single dose of about 1 mg deuterium-labelled acrylamide (d(3)-AA), representing 13 microg/kg body weight, in drinking water. Urine samples before dosing and within 46 h after the dose were analysed for d(3)-AAMA and d(3)-GAMA by LC-ESI-MS/MS. A first phase of increase in urinary concentration was found to last 18 h with a broad plateau between 8 and 18 h for AAMA, and 22 h for GAMA. Elimination half-lives of both AAMA and GAMA were estimated to be approximately 3.5 h for the first phase and more than 10 h up to few days for the second phase. Total recovery in urine after 24 h was about 51% as the sum of AAMA and GAMA and hereby well in accordance with former studies in rats. After 2 days AAMA, accounting for altogether 52% of the total AA dose, was the major metabolite of AA in humans. GAMA, accounting for 5%, appeared as a minor metabolite of AA. In humans we found a urinary ratio of 0.1 for GAMA/AAMA compared to previously reported values of 0.2 for rats and 0.5 for mice. Therefore, the metabolic fate of AA in humans was more similar to that in rats than in mice as already demonstrated in terms of the haemoglobin adducts. Consequently a genotoxic potency of AA mediated by GA could be supposed to be comparable in rats and humans.

A nonlinear dosimetric model for hemoglobin adduct formation by the neurotoxic agent acrylamide and its genotoxic metabolite glycidamide.

Hemoglobin (Hb) adducts, formed by the neurotoxic agent acrylamide (AA) and its genotoxic metabolite glycidamide (GA), were measured in the rat by means of a method for simultaneous determination of the adducts formed to cysteine. A novel, nonlinear dosimetric model was developed to describe Hb adduct formation. This model incorporates the saturable kinetics of the metabolic conversion in vivo of AA to GA. The pharmacokinetic parameters Vmax and Km and the first-order rates of elimination, k1 and k2, for AA and GA from all processes except conversion of AA to GA, were estimated directly from Hb adduct data to 19 M hr-1, 66 microM, 0.21 hr-1, and 0.48 hr-1, respectively. At low concentrations, approximately 60% of AA was metabolized to GA. The nonlinear dosimetric model for adduct formation has potential general applicability in high-to-low-dose extrapolation of genotoxic effects.

A Nonlinear Dosimetric Model for Hemoglobin Adduct Formation by the Neurotoxic Agent Acrylamide and Its Genotoxic Metabolite Glycidamide

Hemoglobin (Hb) adducts, formed by the neurotoxic agent acrylamide (AA) and its genotoxic metabolite glycidamide (GA), were measured in the rat by means of a method for simultaneous determination of the adducts formed to cysteine. A novel, nonlinear dosimetric model was developed to describe Hb adduct formation. This model incorporates the saturable kinetics of the metabolic conversion in vivo of AA to GA. The pharmacokinetic parameters Vmax and Km and the first-order rates of elimination, k1 and k2, for AA and GA from all processes except conversion of AA to GA, were estimated directly from Hb adduct data to 19 M hr-1, 66 microM, 0.21 hr-1, and 0.48 hr-1, respectively. At low concentrations, approximately 60% of AA was metabolized to GA. The nonlinear dosimetric model for adduct formation has potential general applicability in high-to-low-dose extrapolation of genotoxic effects.