Obesity Induces DNA Damage in Mammary Epithelial Cells Exacerbated by Acrylamide Treatment through CYP2E1-Mediated Oxidative Stress.

Obesity and environmental toxins are risk factors for breast cancer; however, there is limited knowledge on how these risk factors interact to promote breast cancer. Acrylamide, a probable carcinogen and obesogen, is a by-product in foods prevalent in the obesity-inducing Western diet. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to the genotoxic epoxide, glycidamide, and is associated with an increased risk for breast cancer. To investigate how acrylamide and obesity interact to increase breast cancer risk, female mice were fed a low-fat (LFD) or high-fat diet (HFD) and control water or water supplemented with acrylamide at levels similar to the average daily exposure in humans. While HFD significantly enhanced weight gain in mice, the addition of acrylamide did not significantly alter body weights compared to respective controls. Mammary epithelial cells from obese, acrylamide-treated mice had increased DNA strand breaks and oxidative DNA damage compared to all other groups. In vitro, glycidamide-treated COMMA-D cells showed significantly increased DNA strand breaks, while acrylamide-treated cells demonstrated significantly higher levels of intracellular reactive oxygen species. The knockdown of CYP2E1 rescued the acrylamide-induced oxidative stress. These studies suggest that long-term acrylamide exposure through foods common in the Western diet may enhance DNA damage and the CYP2E1-induced generation of oxidative stress in mammary epithelial cells, potentially enhancing obesity-induced breast cancer risk.

Reconstructing hepatic metabolic profile and glutathione-mediated metabolic fate of acrylamide.

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA.

A formamidopyrimidine derivative from the deoxyguanosine adduct produced by food contaminant acrylamide induces DNA replication block and mutagenesis.

Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)guanine (dfG), a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis polymerases (Polη, Polι, Polκ, and Polζ) and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-KO cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.

Ultra-processed food consumption and exposure to acrylamide in a nationally representative sample of the US population aged 6 years and older.

Ultra-processed food (UPF) consumption has been associated with cardiovascular disease and cancer. Acrylamide is a probable human carcinogen commonly found in foods that are processed at high temperatures. The aim of this study was to examine the association between dietary energy contribution of UPF and acrylamide exposure, in the US. Among the 4418 participants from cross-sectional 2013-2016 National Health and Nutrition Examination Survey, aged 6+ years, with hemoglobin biomarkers of acrylamide exposure, 3959 that completed the first 24-h dietary recall and had information on all covariates were included in the study. UPF were identified based on the Nova classification system, a four-group food classification based on the extent and purpose of industrial food processing. Linear regression was used to compare average acrylamide and glycidamide hemoglobin (HbAA+HbGA) concentrations across quintiles of daily energy contribution of UPF. Adjusted geometric means of acrylamide and glycidamide hemoglobin concentrations increased monotonically from the lowest to the highest quintile of UPF consumption in the overall population. Compared to the lowest quintile, the highest quintile had 9.1% higher levels of HbAA+HbGA (94.1 vs. 86.3 pmol/g Hb). These positive associations were statistically significant among males and in the young adult population and were largely driven by UPF which are known potential sources of acrylamide. The main effects remained unchanged when excluding current smokers. As both acrylamides and UPF have been previously associated with cardiovascular disease and cancer, our results suggest that acrylamides in UPF may partially explain previously observed links between UPF consumption and these health outcomes.

Relationship between acrylamide and glycidamide hemoglobin adduct levels and osteoarthritis: a NHANES analysis.

Osteoarthritis (OA) is the most prevalent degenerative joint disease, and acrylamide is a chemical produced when foods are processed at high temperatures. Recent epidemiological research linked acrylamide exposure from the diet and environment to a number of medical disorders. However, whether acrylamide exposure is associated with OA is still uncertain. This study was aimed at assessing the relationship between OA and hemoglobin adducts of acrylamide and its metabolite glycidamide (HbAA and HbGA). Data were taken from four cycles of the US NHANES database (2003-2004, 2005-2006, 2013-2014, 2015-2016). Individuals aged between 40 and 84 years who had complete information on arthritic status as well as HbAA and HbGA levels were eligible for inclusion. Univariate and multivariate logistic regression analysis s was performed to determine associations between study variables and OA. Restricted cubic splines (RCS) were used to examine non-linear associations between the acrylamide hemoglobin biomarkers and prevalent OA. A total of 5314 individuals were included and 954 (18%) had OA. After adjusting for relevant confounders, the highest quartiles (vs. lowest) of HbAA (adjusted odds ratio (aOR) = 0.87, 95% confidence interval (CI), 0.63-1.21), HbGA (aOR = 0.82, 95% CI, 0.60-1.12), HbAA + HbGA (aOR = 0.86, 95% CI, 0.63-1.19), and HbGA/HbAA (aOR = 0.88, 95% CI, 0.63--1.25) were not significantly associated with greater odds for OA. RCS analysis revealed that HbAA, HbGA, and HbAA + HbGA levels were non-linearly and inversely associated with OA (p for non-linearity < 0.001). However, the HbGA/HbAA ratio displayed a U-shaped relationship with prevalent OA. In conclusion, acrylamide hemoglobin biomarkers are non-linearly associated with prevalent OA in a general US population. These findings implicate ongoing public health concerns for widespread exposure to acrylamide. Further studies are still warranted to address the causality and biologic mechanisms underlying the association.

Dynamic assessment of the relationship between oxidative stress and apoptotic pathway in embryonic fibroblast cells exposed to glycidamide: possible protective role of hesperidin.

Worldwide research is being conducted to determine the level of acrylamide (ACR) that humans are exposed to from food and environmental sources. Glycidamide (GA) is an important epoxide metabolite of ACR, and its cytotoxicity is stronger than ACR. In this study, it was aimed to elucidate the effects and underlying mechanisms of GA on the induction of apoptosis in embryonic fibroblast cells. The toxicogenomic profile of GA was studied in terms of both apoptotic and oxidative stress. Embryonic fibroblast cells were exposed to GA (1 and 1000 µM) in the presence and absence of hesperidin (Hes) (20 µM) or vitamin C (VitC) (50 µM) for 24 h. Cell viability, cytotoxicity, lipid peroxidation, hydroxyl radicals, hydrogen peroxide, antioxidant enzyme levels and gene expressions, apoptotic, and oxidative stress-related gene expressions were measured in embryonic fibroblast cells. The results showed that GA induced cytotoxicity and diminished the expression levels of apoptotic genes. Furthermore, GA increased the levels of oxidative stress markers and significantly changed the oxidative stress-related gene expression. It has been determined that antioxidant molecules are considerably suppressed in GA-induced toxicity at both gene and enzyme levels. In addition to these results, when VitC, which is known to have strong antioxidant properties in eliminating the toxic effects of GA, is taken as reference, it has been proven that Hes has stronger antioxidant properties compared to VitC. Finally, GA-induced apoptosis in embryonic fibroblast cells is associated with nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress and Hes has antioxidant properties with strong effects.

Acrylamide-induced damage to postsynaptic plasticity is CYP2E1 dependent in an SH-SY5Y co-culture system.

Acrylamide (ACR), a neurotoxic substance, is characterized by a range of industrial and population exposures. The effects of ACR on synapses have been examined, but the regulation and molecular mechanism of key proteins related to ACR and its metabolite glycidamide (GA) have not been elucidated. In this study, we constructed two co-culture systems to mimic neurons that do not express and overexpress CYP2E1. In these co-cultures, we observed the effects and relative influence of ACR and GA on cell survival as well as synaptic structural and functional plasticity. Next, we investigated the relationship between ACR-induced nerve damage and key proteins in the postsynaptic membrane. After ACR exposure, cell death and synaptic damage were significantly worse in CYP2E1-overexpressing co-culture systems, suggesting that ACR-induced neurotoxicity may be related to metabolic efficiency (including CYP2E1 activity). Moreover, with increasing doses of ACR, the key postsynaptic membrane proteins PSD-95 expression was reduced and CaMKII and NMDAR-2B phosphorylation was increased. ACR exposure also triggered a rapid dose- and time-dependent increase in intracellular Ca2+, whose changes can affect the expression of the above-mentioned key proteins. In summary, we clarified the relationship between ACR exposure, neuronal damage and postsynaptic plasticity and proposed an ACR-CYP2E1-GA: Ca2+-PSD-95-NMDAR-Ca2+-CaMKII effect chain. This information will further improve the development of an alternative pathway strategy for investigating the risk posed by ACR.

Trends of Exposure to Acrylamide as Measured by Urinary Biomarkers Levels within the HBM4EU Biomonitoring Aligned Studies (2000-2021).

Acrylamide, a substance potentially carcinogenic in humans, represents a very prevalent contaminant in food and is also contained in tobacco smoke. Occupational exposure to higher concentrations of acrylamide was shown to induce neurotoxicity in humans. To minimize related risks for public health, it is vital to obtain data on the actual level of exposure in differently affected segments of the population. To achieve this aim, acrylamide has been added to the list of substances of concern to be investigated in the HBM4EU project, a European initiative to obtain biomonitoring data for a number of pollutants highly relevant for public health. This report summarizes the results obtained for acrylamide, with a focus on time-trends and recent exposure levels, obtained by HBM4EU as well as by associated studies in a total of seven European countries. Mean biomarker levels were compared by sampling year and time-trends were analyzed using linear regression models and an adequate statistical test. An increasing trend of acrylamide biomarker concentrations was found in children for the years 2014-2017, while in adults an overall increase in exposure was found to be not significant for the time period of observation (2000-2021). For smokers, represented by two studies and sampling for, over a total three years, no clear tendency was observed. In conclusion, samples from European countries indicate that average acrylamide exposure still exceeds suggested benchmark levels and may be of specific concern in children. More research is required to confirm trends of declining values observed in most recent years.

Time Trends of Acrylamide Exposure in Europe: Combined Analysis of Published Reports and Current HBM4EU Studies.

More than 20 years ago, acrylamide was added to the list of potential carcinogens found in many common dietary products and tobacco smoke. Consequently, human biomonitoring studies investigating exposure to acrylamide in the form of adducts in blood and metabolites in urine have been performed to obtain data on the actual burden in different populations of the world and in Europe. Recognizing the related health risk, the European Commission responded with measures to curb the acrylamide content in food products. In 2017, a trans-European human biomonitoring project (HBM4EU) was started with the aim to investigate exposure to several chemicals, including acrylamide. Here we set out to provide a combined analysis of previous and current European acrylamide biomonitoring study results by harmonizing and integrating different data sources, including HBM4EU aligned studies, with the aim to resolve overall and current time trends of acrylamide exposure in Europe. Data from 10 European countries were included in the analysis, comprising more than 5500 individual samples (3214 children and teenagers, 2293 adults). We utilized linear models as well as a non-linear fit and breakpoint analysis to investigate trends in temporal acrylamide exposure as well as descriptive statistics and statistical tests to validate findings. Our results indicate an overall increase in acrylamide exposure between the years 2001 and 2017. Studies with samples collected after 2018 focusing on adults do not indicate increasing exposure but show declining values. Regional differences appear to affect absolute values, but not the overall time-trend of exposure. As benchmark levels for acrylamide content in food have been adopted in Europe in 2018, our results may imply the effects of these measures, but only indicated for adults, as corresponding data are still missing for children.

Two decades of research in dietary acrylamide: What do we know today.

After nearly two decades since acrylamide was first raised as a potential safety issue in foods, significant progress has been made in understanding its formation during cooking, how to reduce levels in the most concerned foods, and the possible cancer risk to humans. Despite the huge wealth of knowledge gathered on this topic over the past years, a few new discoveries in occurrence, mitigation, analysis and risk assessment are worthy to note. This short review highlights the salient novelties pertaining to acrylamide, particularly in the areas of formation & analysis, existing and possible future regulations in the European Union, and finally considerations that may lead to possibly revisiting the toxicity of acrylamide and the main metabolite, glycidamide.

Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure.

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10-8 - 3 ×10-3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10-7 - 1 ×10-8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14-28 days of differentiation. IC50 for cell death of AA and GA was 5.2 × 10-3 M and 5.8 × 10-4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10-7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by ßIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.

Catechins protect against acrylamide- and glycidamide-induced cellular toxicity via rescuing cellular apoptosis and DNA damage.

Acrylamide (AA) occurs in both various environmental and dietary sources and has raised widespread concern as a probable carcinogen. Glycidamide (GA) is the main genotoxic metabolite through P450 2E1 (CYP2E1). In the present study, we investigated the protective effect of (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin (EC) against AA- and GA-induced hepatotoxicity in HepG2 cells. The results demonstrated that EC and EGCG inhibited AA- and GA-induced cytotoxicity and mitochondria-mediated cellular apoptosis. Moreover, exposure to AA (100 µg/mL) and GA (50 µg/mL) caused cell cycle arrest and DNA damage, while EC and EGCG ranging from 12.5 to 50 µg/mL rescued cell cycle arrest and inhibited DNA damage. Furthermore, EC and EGCG down-regulated pro-apoptotic protein Bax and Caspase 3 after a 24-h treatment in HepG2 cells exposed to AA (100 µg/mL) or GA (50 µg/mL). Also, the intervention with EC or EGCG up-regulated the expression of DNA repair related protein PARP and down-regulated the expression of Cleaved-PARP. Besides, EC exerted better protective effect than EGCG against AA- and GA-induced cytotoxicity in HepG2 cells. Altogether, EC and EGCG were effective in protecting AA- and GA-induced hepatotoxicity via rescuing cellular apoptosis and DNA damage, as well as promoting cell cycle progression in HepG2 cells.

Glycidamide and cis-2-butene-1,4-dial (BDA) as potential carcinogens and promoters of liver cancer - An in vitro study.

Acrylamide and furan are environmental and food contaminants that are metabolized by cytochrome P450 2E1 (CYP2E1), giving rise to glycidamide and cis-2-butene-1,4-dial (BDA) metabolites, respectively. Both glycidamide and BDA are electrophilic species that react with nucleophilic groups, being able to introduce mutations in DNA and perform epigenetic remodeling. However, whereas these carcinogens are primarily metabolized in the liver, the carcinogenic potential of acrylamide and furan in this organ is still controversial, based on findings from experimental animal studies. With the ultimate goal of providing further insights into this issue, we explored in vitro, using a hepatocyte cell line and a hepatocellular carcinoma cell line, the putative effect of these metabolites as carcinogens and cancer promoters. Molecular alterations were investigated in cells that survive glycidamide and BDA toxicity. We observed that those cells express CD133 stemness marker, present a high proliferative capacity and display an adjusted expression profile of genes encoding enzymes involved in oxidative stress control, such as GCL-C, GSTP1, GSTA3 and CAT. These molecular changes seem to be underlined, at least in part, by epigenetic remodeling involving histone deacetylases (HDACs). Although more studies are needed, here we present more insights towards the carcinogenic capacity of glycidamide and BDA and also point out their effect in favoring hepatocellular carcinoma progression.

Maternal acrylamide exposure during pregnancy and fetal growth: A systematic review and dose-response meta-analysis of epidemiological studies.

BACKGROUND: Acrylamide is a food contaminant linked to developmental toxicity in animals and possibly in humans. OBJECTIVES: We performed a systematic review and dose-response meta-analysis of epidemiological studies evaluating the relationship between maternal acrylamide exposure during pregnancy and the risk of being small for gestational age (SGA) and birth weight, birth head circumference and birth length. METHODS: We performed the literature search in PubMed, Scopus, and Web of Science, until June 6th, 2022. Studies carried out in mother-newborn pairs, assessing maternal acrylamide exposure during pregnancy, either via dietary assessments or biomarkers i.e., hemoglobin adducts of acrylamide (AA-Hb) and glycidamide (GA-Hb), and evaluating birth outcomes were included. We employed a random-effects model to assess the pooled effect estimates and their 95% confidence intervals (CI) for the association between acrylamide exposure and birth outcomes. Risk of Bias for Nutrition Observational Studies tool was used for bias assessment. RESULTS: Out of 169 records identified, five original studies were eligible, including 53,870 mother-newborn pairs in total. Means were 21.9 µg/day for estimated dietary acrylamide exposure (3 studies), and 18.4 and 14.9 pmol/g for AA-Hb and GA-Hb, respectively (2 studies). Higher risk of SGA and lower birth weight and head circumference were observed in the highest quartile of AA-Hb [odds ratio (OR): 1.20 (95% CI: 1.08; 1.33); mean difference (MD): -131 g (95% CI: -204; -58) and -0.31 cm (95% CI: -0.58; -0.04), respectively], and GA-Hb [OR: 1.36 (95% CI: 1.13; 1.64), MD: -161 g (95% CI: -271; -52); and MD: -0.38 cm (95% CI: -0.66; -0.10), respectively], whereas a lower birth length was observed only in the highest quartile of GA-Hb (MD: -0.85 cm (95% CI: -1.38; -0.33). Results from the dose-response meta-analysis between increasing maternal acrylamide exposure during pregnancy and birth weight showed no clear evidence of a deviation from linearity. CONCLUSIONS: Overall, our findings strengthen the evidence of an adverse effect of maternal acrylamide exposure during pregnancy on fetal growth. These results encourage to increase preventive actions towards lowering acrylamide exposure in the population.

Assessment of the genotoxicity of acrylamide.

EFSA was requested to deliver a statement on a recent publication revisiting the evidence for genotoxicity of acrylamide (AA). The statement was prepared by a Working Group and was endorsed by the CONTAM Panel before its final approval. In interpreting the Terms of Reference, the statement considered the modes of action underlying the carcinogenicity of AA including genotoxic and non-genotoxic effects. Relevant publications since the 2015 CONTAM Panel Opinion on AA in food were reviewed. Several new studies reported positive results on the clastogenic and mutagenic properties of AA and its active metabolite glycidamide (GA). DNA adducts of GA were induced by AA exposure in experimental animals and have also been observed in humans. In addition to the genotoxicity of AA, there is evidence for both secondary DNA oxidation via generation of reactive oxygen species and for non-genotoxic effects which may contribute to carcinogenesis by AA. These studies extend the information assessed by the CONTAM Panel in its 2015 Opinion, and support its conclusions. That Opinion applied the margin of exposure (MOE) approach, as recommended in the EFSA Guidance for substances that are both genotoxic and carcinogenic, for risk characterisation of the neoplastic effects of AA. Based on the new data evaluated, the MOE approach is still considered appropriate, and an update of the 2015 Opinion is not required at the present time.

Analysis of Acrylamide and Glycidamide in Dried Blood Spot of Smokers Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

INTRODUCTION: Acrylamide is a genotoxic substance that can be found in cigarette smoke. Acrylamide is metabolized by the CYP2E1 enzyme in the body to form glycidamides, an epoxide that is reactive to DNA and can form carcinogenic adducts. Therefore, exposure to acrylamide can potentially cause cancer. This study aims to analyze the levels of acrylamide and glycidamide in dried blood spot samples of smokers using propanamide as an internal standard and non-smokers as the control subjects. METHODS: Dried blood spot samples were extracted using the protein precipitation method and then analyzed by liquid chromatography-tandem mass spectrometry. Mass detection was performed using positive type electro spray ionization and multiple reaction monitoring type with m/z 72.0>55.02 for acrylamide, 88.1>45.0 for glycidamide, and 74.0>57.1 for propanamide as the internal standard. RESULTS: Acrylamide and glycidamide levels in the dried blood spot sample of smokers ranged between 3.91 -10.25 µg/mL and 1.006-3.58 µg/mL, respectively. Data of the non-smokers on acrylamide and glycidamide levels were 0.75-3.16 µg/mL and 0-0.91 µg/mL. DISCUSSION: The significant value of acrylamide and glycidamide between smokers and non-smokers was p < 0.05, which showed that there is a significant difference between acrylamide and glycidamide concentration in smokers and non-smoker subjects. The results of this study suggest that dried blood spots can be used to determine acrylamide and glycidamide levels in humans. Theoretically, acrylamide and glycidamide concentration should correlate to each other; however in reality, there are other factors (such as CYP2E1 polymorphism, dietary intake, etc) that can cause variation in their respective concentration.

Total cholesterol: a potential mediator of the association between exposure to acrylamide and hypertension risk in adolescent females.

Acrylamide (AA) exposure is associated with a range of adverse health effects. However, whether AA exposure is related to hypertension in adolescents remains unclear. The associations of blood hemoglobin biomarkers of AA (HbAA) and its metabolite glycidamide (HbGA) with hypertension risk, diastolic blood pressure (DBP), and systolic blood pressure (SBP) were evaluated by multivariate logistic regression and linear regression. We identified a potential positive association between blood HbGA and hypertension risk in adolescent females (OR 1.81, 95% CI 1.00-3.30; P for trend = 0.022); however, there was no correlation in the non-linear model (P = 0.831). In the sex-stratified linear models, blood HbGA level had a strong positive association with SBP in adolescent females (beta 0.84, 95% CI 0.13-1.55, P = 0.020). Mechanistically, a one-unit increase in blood HbGA (ln transformed) was associated with a 2.83 mg/dL increase in total cholesterol (TC) among females in the fully adjusted model. Mediation analysis showed that TC mediated 24.15% of the association between blood HbGA level and the prevalence of hypertension in females. The present results provide epidemiological evidence that exposure to AA, mainly its metabolite glycidamide, is positively associated with the prevalence of hypertension or increased SBP in adolescent females.

The construction and application of physiologically based toxicokinetic models for acrylamide, glycidamide and their biomarkers in rats and humans.

Acrylamide (AA), a class 2A probable carcinogen to humans classified by the International Agency for Research on Cancer, has attracted extensive attention worldwide since it was widely used in industrial and domestic water treatment and detected in thermal processing foods. The metabolic adducts of AA and its primary metabolite glycidamide (GA) have been served as biomonitoring markers of AA intake, but the physiologically based toxicokinetics (PBTK) models to estimate internal dosimetry still remain unclear. An updated PBTK model for AA, GA and their metabolic biomarkers in rats and humans was developed and extended with time-course datasets from both literatures and our experiments. With adjustments to the model parameters, linear regression correlation coefficient (R2) between the fitting values and the validation datasets of rats and humans was greater than 0.76. The current model fits well with the experimental datasets of urinary N-acetyl-S-(2-carbamoylethyl)-l-cysteine (AAMA) and (N-(R,S)-acetyl-S-(carbamoyl-2-hydroxyethyl)-l-cysteine) (GAMA) of rats exposed to AA from 0.1 to 50 mg/kg b.w. and humans exposed to AA from 0.0005 to 0.020 mg/kg b.w., indicating the robustness of the current models. Parameters for adduct of AA with N-terminal valine of hemoglobin (AAVal) were extended to humans and validated. Kinetic parameters for rats were assessed and validated based upon fit to the experimental datasets for liver N3-(2-carbamoyl-2-hydroxyethyl)-adenine (N3-GA-Ade) and N7-(2-carbamoyl-2-hydroxyethyl)-guanine (N7-GA-Gua) adducts. Compared with the previous model, the developed model included the correlation between AA intake and its mercapturic acid adducts, AAMA and GAMA, in a larger dose range with new experimental data, and parameters for AAVal, N3-GA-Ade and N7-GA-Gua were improved and verified. The current multi-component PBTK models provide a superior foundation for the estimation of short-term to medium and long-term intake levels of human exposure to AA.

Simultaneous toxicokinetics characterization of acrylamide and its primary metabolites using a novel microdialysis isotope-dilution liquid chromatography mass spectrometry method.

Acrylamide (AA) is a toxicant in high-temperature processed foods and an animal carcinogen. Upon absorption, AA is metabolized to glycidamide (GA) or conjugates with glutathione (AA-GSH). Important advantages of microdialysis coupled with liquid chromatography-tandem mass spectrometry (MD-LC-MS/MS) include its minimization of potential losses during sample collection, storage and preparation, as well as an improvement in temporal resolution for toxicokinetics (TKs). We aimed to simultaneously study the TKs of AA and products of its primary metabolism using an isotope-dilution (ID) MD-LC-MS/MS method. MD probes implanted into the jugular vein/right atrium of anesthetized Sprague Dawley rats were connected to the ID-LC-MS/MS for continuous monitoring of AA, GA and AA-GSH in the blood every 15 min over 8 h following intraperitoneal AA administration (0.1 mg/kg or 5 mg/kg). AA, GA, and AA-GSH TKs followed linear kinetics: GA AUC/AA AUC = 0.11 and AA-GSH AUC/AA AUC = 0.011 at 5 mg/kg. Elimination half-life (Te1/2) values were 2.44 ± 0.70, 4.93 ± 2.37 and 3.47 ± 1.47 h for AA, GA and AA-GSH, respectively. GA TKs reached a plateau at 3-6 h, suggesting that metabolic saturation of AA and Te1/2 values of the analytes were prolonged with AA at 5 mg/kg. Our results demonstrate that oxidation of AA to GA overwhelmed the conjugation of AA with GSH. Our innovative MD-ID-LC-MS/MS method facilitates the simultaneous characterization of multiple TKs associated with toxicants and their active metabolites with excellent temporal resolution to capture metabolic saturation of AA to GA.

A Benchmark analysis of acrylamide-derived DNA adducts in rat hepatocytes in culture measured by a new, highly sensitive method.

Acrylamide (AA) is a carcinogen formed during thermal food processing and can cause tumors in rodents while its carcinogenic potency in humans is unclear. Metabolism of AA, preferentially in the liver, leads to glycidamide (GA) forming N7-GA-guanine (N7-GA-Gua) as the major AA-derived DNA adduct in rodents. Here, a novel method allowing high sensitivity by avoidance of major matrix effects was applied to analyze N7-GA-Gua levels in nuclear DNA from rat hepatocytes in primary culture. We could thus for the first time detect a background level of 5-10 adducts/108 nucleosides in untreated hepatocytes. Incubation with AA did not result in a statistically significant increase in adduct levels over background up to a substrate concentration of 500 µM although a trend to slightly higher adduct levels was observed at and above 200 µM AA. At concentrations > 500 µM significant increases in N7-GA-Gua levels were found. When Benchmark concentration (BMC) modeling was applied to the data, non-linear concentration-response curves were obtained suggesting that AA started to cause measurable increases over background of N7-GA-Gua levels above certain concentrations only. Calculation of the composite BMCL10 (Lower Bound of a 95 % confidence interval) of a BMC leading to a 10 % increase of N7-GA-Gua levels over background resulted in a value of 6.35 µM AA after 24 h. A concentration below this value cannot be expected to lead to an increase in N7-GA-Gua of more than 10 % over the background seen in untreated hepatocytes.