Association between acrylamide exposure and sex hormones among premenopausal and postmenopausal women: NHANES, 2013-2016.

PURPOSE: Acrylamide (AA) is a potential carcinogen that mainly comes from fried, baked and roasted foods, and Hb adducts of AA (HbAA) and its metabolite glycidamide (HbGA) are the biomarkers of its exposure. Increasing evidence suggests that AA is associated with various hormone-related cancers. This study aims to explore the association of HbAA and HbGA with female serum sex hormone concentrations. METHODS: 942 women from the National Health and Nutrition Examination Survey cycles (2013-2016) were included in this cross-sectional study. The associations between HbAA or HbGA or HbGA/HbAA and sex hormones were assessed by the multiple linear regression. Further stratified analyses were conducted to figure out the effects of menopausal status, BMI and smoking status on sex hormone levels. RESULTS: Among all participants, 597 were premenopausal and 345 were postmenopausal. HbAA was positively associated with both two androgen indicators. Specifically, a ln-unit increase in HbAA was associated with 0.41 ng/dL higher ln(total testosterone, TT) (95% CI 0.00, 0.27) and 0.14 ng/dL higher ln(free testosterone) (95%CI 0.00, 0.28), respectively. However, HbGA concentrations had no association with sex hormones in the overall population. Additionally, HbGA/HbAA was negatively associated with TT and SHBG in the overall population as well as postmenopausal women. In stratified analysis, higher HbAA was associated with rising TT in postmenopausal women (ß = 0.29, 95%CI 0.04, 0.53) and underweight/normal-weight women (ß = 0.18, 95%CI 0.03, 0.33). Other indicators had no significant association detected in estradiol and sex hormone-binding globulin. CONCLUSION: Our results revealed that HbAA was positively associated with androgen concentrations, especially in postmenopausal and BMI < 25 women.

Chronotoxicity of Acrylamide in Mice Fed a High-Fat Diet: The Involvement of Liver CYP2E1 Upregulation and Gut Leakage.

Acrylamide (ACR) is produced under high-temperature cooking of carbohydrate-rich foods via the Maillard reaction. It has been reported that ACR has hepatic toxicity and can induce liver circadian disorder. A high fat diet (HFD) could dysregulate liver detoxification. The current study showed that administration of ACR (100 mg/kg) reduced the survival rate in HFD-fed mice, which was more pronounced when treated during the night phase than during the day phase. Furthermore, ACR (25 mg/kg) treatment could cause chronotoxicity in mice fed a high-fat diet, manifested as more severe mitochondrial damage of liver during the night phase than during the day phase. Interestingly, HFD induced a higher CYP2E1 expressions for those treated during the night phase, leading to more severe DNA damage. Meanwhile, the expression of gut tight junction proteins also significantly decreases at night phase, leading to the leakage of LPSs and exacerbating the inflammatory response at night phase. These results indicated that a HFD could induce the chronotoxicity of ACR in mice liver, which may be associated with increases in CYP2E1 expression in the liver and gut leak during the night phase.

Determination of N7-glycidamide guanine adducts in human blood DNA following exposure to dietary acrylamide using liquid chromatography/tandem mass spectrometry.

RATIONALE: Acrylamide is classified as a probable human carcinogen that is metabolised to glycidamide, which can covalently bind to DNA. The aim of this study was to investigate the formation of N7-glycidamide guanine (N7-GA-Gua) adducts in human blood DNA following exposure to acrylamide present in carbohydrate-rich foods as part of the normal human diet. METHODS: Lymphocyte DNA was extracted from blood samples obtained from healthy human volunteers. Following thermal depurination of the DNA samples, N7-GA-Gua adducts were quantified using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method incorporating a stable isotope labelled internal standard. Estimated dietary acrylamide intake was recorded by completion of food frequency questionnaires for the 24 hours prior to volunteer blood donation. RESULTS: An LC/MS/MS method was validated with a limit of detection of 0.25 fmol and a lower limit of quantitation of 0.50 fmol on column. N7-GA-Gua adducts were detected in human blood DNA with the levels ranging between 0.3 to 6.3 adducts per 108 nucleotides. The acrylamide intake was calculated from the food frequency questionnaires ranging between 20.0 and 78.6 µg. CONCLUSIONS: Identification and quantification of N7-GA-Gua adducts in the blood DNA of healthy volunteers suggests that dietary acrylamide exposure may lead to the formation of DNA adducts. This important finding warrants further investigation to ascertain a correlation between environmental/dietary acrylamide exposure and levels of DNA adducts.

Evaluation of acrylamide production by Rhodococcus rhodochrous (RS-6) cells immobilized in agar matrix.

AIMS: The efficiency of acrylamide production was examined with immobilized cells of Rhodococcus rhodochrous (RS-6) containing NHase. METHODS AND RESULTS: Different entrapment matrices such as agar, alginate and polyacrylamide were used. Various immobilization parameters like agar concentration, cell concentration and reaction conditions affecting the bioconversion process using suitable matrices were determined. The cells immobilized with agar matrix were found to be most effective for acrylonitrile conversion. The bioconversion was more efficient in beads prepared with 2% agar and 5% (v/v) cell concentration. The entire conversion of acrylonitrile to acrylamide with agar entrapped cells was achieved in 120 min at 15°C. The agar entrapped R. rhodochrous (RS-6) cells exhibited 8% (w/v) tolerance to acrylonitrile and 35% tolerance to acrylamide. The immobilized cells also retained 50% of its conversion ability up to seven cycles. The laboratory-scale (1 L) production resulted in 466 g L-1 accumulation of acrylamide in 16 h. CONCLUSIONS: The cells immobilized in agar showed better stability and biocatalytic properties and increased reusability potential. SIGNIFICANCE AND IMPACT OF THE STUDY: The agar-immobilized Rhodococcus rhodochrous (RS-6) cells showed enhanced tolerance for both the substrate and product and is economical for the large-scale production of acrylamide.

Appraisal of cytotoxicity and acrylamide mitigation potential of L-asparaginase SlpA from fish gut microbiome.

L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. It has application in the treatment of acute lymphoblastic leukemia in children, as well as in other malignancies, in addition to its role as a food processing aid for the mitigation of acrylamide formation in the baking industry. Its use in cancer chemotherapy is limited due to problems such as its intrinsic glutaminase activity and associated side effects, leading to an increased interest in the search for novel L-asparaginases without L-glutaminase activity. This study reports the cloning and expression of an L-asparaginase contig obtained from whole metagenome shotgun sequencing of Sardinella longiceps gut microbiota. Purified recombinant glutaminase-free L-asparaginase SlpA was a 74 kDa homodimer, with maximal activity at pH 8 and 30 °C. Km and Vmax of SlpA were determined to be 3.008 mM and 0.014 mM/min, respectively. SlpA displayed cytotoxic activity against K-562 (chronic myeloid leukemia) and MCF-7 (breast cancer) cell lines with IC50 values of 0.3443 and 2.692 U/mL, respectively. SlpA did not show any cytotoxic activity against normal lymphocytes and was proved to be hemocompatible. Pre-treatment of biscuit and bread dough with different concentrations of SlpA resulted in a clear, dose-dependent reduction of acrylamide formation during baking. KEY POINTS: • Cloned and expressed L-asparaginase (SlpA) from fish gut microbiota • Purified SlpA displayed good cytotoxicity against K-562 and MCF-7 cell lines • SlpA addition caused a significant reduction of acrylamide formation during baking.

Acrylamide and Potential Risk of Diabetes Mellitus: Effects on Human Population, Glucose Metabolism and Beta-Cell Toxicity.

Diabetes mellitus is a frequent endocrine disorder characterized by hyperglycemia. Acrylamide (AA) is food contaminant formed during the high-temperature processing of food rich in carbohydrates and low in proteins. Recent human epidemiological studies have shown a potential association between AA exposure and the prevalence of diabetes in the general population. In male rats, AA treatment promoted pancreatic islet remodeling, which was determined by alpha-cell expansion and beta-cell reduction, while in female rats AA caused hyperglycemia and histopathological changes in pancreatic islets. In vitro and in vivo rodent model systems have revealed that AA induces oxidative stress in beta cells and that AA impairs glucose metabolism and the insulin signaling pathway. Animal studies have shown that diabetic rodents are more sensitive to acrylamide and that AA aggravates the diabetic state. In this review, we provide an overview of human epidemiological studies that examined the relation between AA exposure and glucose disorders. In addition, the effects of AA treatment on pancreatic islet structure, beta-cell function and glucose metabolism in animal models are comprehensively analyzed with an emphasis on sex-related responses. Furthermore, oxidative stress as a putative mechanism of AA-induced toxicity in beta cells is explored. Finally, we discuss the effects of AA on diabetics in a rodent model system.

Effect of Acrylamide Treatment on Cyp2e1 Expression and Redox Status in Rat Hepatocytes.

Acrylamide (AA) toxicity is associated with oxidative stress. During detoxification, AA is either coupled to gluthatione or biotransformed to glycidamide by the enzyme cytochrome P450 2E1 (CYP2E1). The aim of our study was to examine the hepatotoxicity of AA in vivo and in vitro. Thirty male Wistar rats were treated with 25 or 50 mg/kg b.w. of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), CYP2E1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 expression in liver was carried out. Bearing in mind that the liver is consisted mainly of hepatocytes, in a parallel study, we used the rat hepatoma cell line H4IIE to investigate the effects of AA at IC20 and IC50 concentrations on the redox status and the activity of CAT, SOD, and glutathione-S-transferase (GST), their gene expression, and CYP2E1 and iNOS expression. Immunohistochemically stained liver sections showed that treatment with AA25mg induced a significant decrease of CYP2E1 protein expression (p < 0.05), while treatment with AA50mg led to a significant increase of iNOS protein expression (p < 0.05). AA treatment dose-dependently elevated SOD2 protein expression (p < 0.05), while SOD1 protein expression was significantly increased only at AA50mg (p < 0.05). CAT protein expression was not significantly affected by AA treatments (p > 0.05). In AA-treated H4IIE cells, a concentration-dependent significant increase in lipid peroxidation and nitrite levels was observed (p < 0.05), while GSH content and SOD activity significantly decreased in a concentration-dependent manner (p < 0.05). AA IC50 significantly enhanced GST activity (p < 0.05). The level of mRNA significantly increased in a concentration-dependent manner for iNOS, SOD2, and CAT in AA-treated H4IIE cells (p < 0.05). AA IC50 significantly increased the transcription of SOD1, GSTA2, and GSTP1 genes (p < 0.05), while AA IC20 significantly decreased mRNA for CYP2E1 in H4IIE cells (p < 0.05). Obtained results indicate that AA treatments, both in vivo and in vitro, change hepatocytes; drug-metabolizing potential and disturb its redox status.

A DFT Study of the Reaction of Acrylamide with L-Cysteine and L-Glutathione.

Thermal processing of certain foods implies the formation of acrylamide, which has been proven to provoke adverse effects on human health. Thus, several strategies to mitigate it have been developed. One of them could be the application of organosulfur compounds obtained from natural sources to react with the acrylamide, forming non-toxic adducts. A DFT study of the acrylamide reaction with the organosulfur model compounds L-cysteine and L-glutathione by Michael addition and a free radical pathway complemented by a kinetic study of these model molecules has been applied. The kinetic evaluation results demonstrate that the L-glutathione reaction exhibited a higher rate constant than the other studied compound.

Protective Effect of Lycium ruthenicum Polyphenols on Oxidative Stress against Acrylamide Induced Liver Injury in Rats.

Acrylamide (ACR) is formed during tobacco and carbohydrate-rich food heating and is widely applied in many industries, with a range of toxic effects. The antioxidant properties of Lycium ruthenicum polyphenols (LRP) have been established before. This study aimed to research the protective effect of LRP against ACR-induced liver injury in SD rats. Rats were divided into six groups: Control, ACR (40 mg/kg/day, i.g.), LRP (50, 100, and 200 mg/kg/day, i.g.) plus ACR, and LRP groups. After 19 days, we evaluated oxidative status and mitochondrial functions in the rat's liver. The results showed that glutathione (GSH) and superoxide dismutase (SOD) levels increased after LRP pretreatment. In contrast, each intervention group reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels compared to the ACR group. Meanwhile, alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver mitochondrial ATPase activity, mRNA expression of mitochondrial complex I, III, and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and its downstream proteins were all increased. This study suggested that LRP could reduce ACR-induced liver injury through potent antioxidant activity. LRP is recommended as oxidative stress reliever against hepatotoxicity.

Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes.

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much-improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity-based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.

Characterization of Penicillium crustosum L-asparaginase and its acrylamide alleviation efficiency in roasted coffee beans at non-cytotoxic levels.

This work aims at isolating a fungal source for L-asparaginase production to be applied in reducing acrylamide levels in coffee beans at non-cytotoxic levels. An L-asparaginase-producing fungus was isolated from an agricultural soil sample and identified as Penicillium crustosum NMKA 511. A maximum L-asparaginase activity of 19.10 U/mL was obtained by the above-mentioned fungus when grown under optimum conditions (i.e. 16.96 g/L sucrose as carbon source, 1.92 g/L peptone as nitrogen source, pH 7.7 and 33.5 °C). Further, the produced L-asparaginase was purified and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that P. crustosum L-asparaginase was a heterodimer enzyme with molecular weights of approximately 41.3 and 44.4 kDa. Also, the purified P. crustosum L-asparaginase was specific towards L-asparagine and showed negligible and no effects towards L-glutamine and D-asparagine, respectively. Additionally, the purified L-asparaginase reduced the acrylamide levels by 80.7% and 75.8% in light and dark roasted coffee beans, respectively. The amount of L-asparaginase used to reduce acrylamide was considered safe when cell viability reached 94.6%.

Associations of acrylamide with non-alcoholic fatty liver disease in American adults: a nationwide cross-sectional study.

BACKGROUND: Acrylamide (AA) is a toxicant to humans, but the association between AA exposure and the risk of non-alcoholic fatty liver disease (NAFLD) remains unclear. In this study, our objective is to examine the cross-sectional association between AA exposure and the risk of NAFLD in American adults. METHODS: A total of 3234 individuals who took part in the National Health and Nutrition Examination Survey (NHANES) 2003-2006 and 2013-2016 were enrolled in the study. NAFLD was diagnosed by the U.S. Fatty Liver Index. Multivariable logistic regression models were applied to estimate the association between AA and NAFLD in the whole group and the non-smoking group. RESULTS: We discovered that in the whole group, serum hemoglobin adducts of AA (HbAA) were negatively associated with the prevalence of NAFLD after adjustment for various covariables (P for trend < 0.001). Compared with individuals in the lowest HbAA quartiles, the odds ratios (ORs) with 95% confidence intervals (CIs) in the highest HbAA quartiles were 0.61 (0.46-0.81) and 0.57 (0.36-0.88) in the whole group and the non-smoking group, respectively. In contrast, HbGA/HbAA showed a significantly positive correlation with the prevalence of NAFLD in both groups (P for trend < 0.001). In addition, HbGA was not significantly associated with NAFLD in the whole group or the non-smoking group. CONCLUSIONS: HbAA is negatively associated with NAFLD whereas HbGA/HbAA is positively associated with NAFLD in adults in the U.S. Further studies are needed to clarify these relationships.

Cord blood acrylamide levels and birth size, and interactions with genetic variants in acrylamide-metabolising genes.

BACKGROUND: Up to now, 3 epidemiological studies have shown clear inverse associations between prenatal acrylamide exposure and birth size. In addition to studying the association between acrylamide and birth size, we investigated the interaction between acrylamide and polymorphisms in acrylamide-metabolising genes, with the aim of probing the causality of the inverse relationship between acrylamide and fetal growth. METHODS: We investigated the association between prenatal acrylamide exposure (acrylamide and glycidamide hemoglobin adduct levels (AA-Hb and GA-Hb) in cord blood) and birth weight, length and head circumference in 443 newborns of the ENVIRONAGE (ENVIRonmental influence ON AGEing in early life) birth cohort. In addition, we studied interaction with single nucleotide polymorphisms (SNPs) in CYP2E1, EPHX1 and GSTP1, using multiple linear regression analysis. RESULTS: Among all neonates, the body weight, length and head circumference of neonates in the highest quartile was - 101 g (95% CI: - 208, 7; p for trend = 0.12), - 0.13 cm (95% CI: - 0.62, 0.36; p for trend = 0.69) and - 0.41 cm (- 0.80, - 0.01; p for trend = 0.06) lower, respectively, compared to neonates in the lowest quartile of AA-Hb in cord blood, For GA-Hb, the corresponding effect estimates were - 222 g (95% CI: - 337, - 108; p for trend = 0.001), - 0.85 (95% CI: - 1.38, - 0.33; p for trend = 0.02) and - 0.55 (95% CI: - 0.98, - 0.11; p for trend = 0.01), respectively. The associations for GA-Hb were similar or stronger in newborns of non-smoking mothers. There was no statistically significant interaction between acrylamide exposure and the studied genetic variations but there was a trend of stronger inverse associations with birth weight and head circumference among newborns with homozygous wildtypes alleles for the CYP2E1 SNPS and with variant alleles for a GSTP1 SNP (rs1138272). CONCLUSIONS: Prenatal dietary acrylamide exposure, specifically in the form of its metabolite glycidamide, was inversely associated with birth weight, length and head circumference. The interaction pattern with SNPs in CYP2E1, although not statistically significant, is an indication for the causality of this association. Other studies are needed to corroborate this finding.

Evaluation of acrylamide-removing properties of bacterial consortia under simulated gastrointestinal conditions.

BACKGROUND: Previous studies have demonstrated the acrylamide-removing properties of probiotic monocultures; however, potential advantages of consortia over monocultures in reducing the dietary exposure to acrylamide have not been proven. Hence this work aims to assess the acrylamide (AA)-binding properties of bacterial consortia, consisting of either probiotic strains and / or representative bacteria of duodenal microbiota, exposed to simulated gastrointestinal conditions (SGC). The AA binding capacity of ten probiotic strains (PS) and six duodenal strains (NDS) was evaluated under different conditions; then, three different consortia (PS, NDS, and PS + NDS) were assessed under SGC. RESULTS: Among individual PS, Bacillus coagulans GBI-30, Lactobacillus fermentum J23, L. pentosus J37 and J24, and L. casei Shirota, exhibited the highest AA-binding capacity (80-87%), while Bifidobacterium catenulatun ATCC27676, Streptococcus salivarius subsp. thermophilus ATCC19258, and S. gallolyticus ATCC9809 were the best (ca. 68%) NDS monocultures. Probiotic strain consortia showed higher (P < 0.05) AA binding capacity (> 90%) than monoculture bacteria. Conversely, individual NDS cultures displayed higher (P < 0.05) binding capacity than NDS consortia (60%). A significant reduction (P < 0.05) in AA removal capacity was observed when consortia were exposed to SGC, PS consortia being the most effective (> 60% removal). CONCLUSION: These results suggest that consortia of specific PS could play an important role in reducing the intestinal availability of acrylamide. © 2021 Society of Chemical Industry.

An Affinity-Based, Cysteine-Specific ATP Analog for Kinase-Catalyzed Crosslinking.

Kinases mediate cell signaling pathways by catalyzing protein phosphorylation. Irregularities in kinase activity are directly associated with disease conditions. Therefore, methods to identify substrates of a particular kinase are needed to understand signaling cascades in normal and diseased states. Photocrosslinking ATP analogs provide powerful tools to study kinases by covalently linking kinases with substrates. However, the involvement of UV light and nonspecific reactivity of current ATP-photocrosslinkers challenge kinase-substrate identification. We report here an affinity-based crosslinking ATP analog, ATP-methylacrylamide (ATP-MAc), that contains a cysteine-reactive acrylamide crosslinking group, which avoids the UV irradiation and non-specific reactivity of prior analogs. Using in vitro kinase assays, ATP-MAc acts as a kinase co-substrate and covalently crosslinks only kinases containing cysteines in the active site. ATP-MAc was also able to crosslink cellular proteins in lysates, documenting compatibility with cell-based studies.

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH.

Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.

A urinary metabolomic study from subjects after long-term occupational exposure to low concentration acrylamide using UPLC-QTOF/MS.

Because long-term occupational exposure to low concentrations of acrylamide (ACR) has the potential to cause neurological damage, it is important to identify biomarkers that can be used to evaluate this risk. In the present study, urine metabolomics of the ACR-exposed and non-exposed groups to identify potential metabolites was carried out using ultra high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. Serum biochemical indexes of the exposed and non-exposed groups were also determined. Principal component analysis showed a differential separation between exposed group and non-exposed group and a total of 7 metabolites were identified in positive and negative ionization modes; Area under curve of anthranilic acid, ß-guanidinopropionic acid and mesobilirubinogen were 0.980, 0.843 and 0.801 respectively and these metabolites showed high sensitivity and specificity. The 13 biochemical indexes were divided into three classes based on physiological functions. Only biomarkers of dysregulated liver function including alanine aminotransferase, aspartic transaminase, total bilirubin, direct bilirubin and triglyceride were significantly higher in the exposed group than in the non-exposed group. This study identifies important related metabolic changes in the bodies of workers after long-term occupational exposure to low concentration ACR and suggests new biomarkers of nervous system injury caused by ACR. The study also provides a sound basis for exploring the biochemical mechanisms and metabolic pathways of nervous system toxicity caused by ACR.

In vitro mutagenicity of selected environmental carcinogens and their metabolites in MutaMouse FE1 lung epithelial cells.

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide -S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide -S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP -S9, 4-ABP ±S9 and N-OH-4-ABP -S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

Acrylamide in human diet, its metabolism, toxicity, inactivation and the associated European Union legal regulations in food industry.

Nowadays acrylamide is known not only as synthetic material used in industry, but also as carcinogenic, cyto- and genotoxic compound which forms during heat-induced process (due to Maillard reaction) mostly in foodstuff such as potato, bakery, plant derivatives products and coffee. The International Agency for Research on Cancer in 1994 declared acrylamide as a probable carcinogenic agent in humans. After metabolic process, acrylamide is distributed to all organs and tissues in human body. Acrylamide is classified as human neurotoxin, because this effect was observed in humans occupationally exposed to this compound. Acrylamide was found to cause apoptosis by mitochondrial dysfunction. Methods of acrylamide inactivation by microorganisms and bioactive diet compounds have also been reviewed. Moreover, there is still deficit of the European Union legal regulation concerning acrylamide mitigation strategies in food. Regulation 2017/2158 from 20 November 2017 is a step in the right direction when it comes to ensuring food safety and maximum levels of acrylamide in foodstuffs, however when exceeding those, it should result in elimination of such food from the market.

Advances in acrylamide bioproduction catalyzed with Rhodococcus cells harboring nitrile hydratase.

Acrylamide is an important bulk chemical used for producing polyacrylamide, which is widely applied in diverse fields, such as enhanced oil recovery and water treatment. Acrylamide production with a superior biocatalyst, free-resting Rhodococcus cells containing nitrile hydratase (NHase), has been proven to be simple but effective, thereby becoming the main method adopted in industry to date. Under the harsh industrial conditions, however, NHase-containing Rhodococcus cells in a natural state are prone to deactivation. Thus, multiple genetic strategies able to evolve recombinant Rhodococcus biocatalysts at either the enzyme or cell level have been reported. While most of the methods on enzyme engineering concentrate on NHase stability enhancement by strengthening the flexible sites, Rhodococcus cell engineering with various methods can enhance both the NHase activity and stability as well. Developing some new types of reactors, especially the microreactor, is also an effective way to improve the hydration process efficiency. Compared with the conventional stirred tank reactor, the membrane dispersion microreactor can enhance the heat and mass transfer in the hydration process with Rhodococcus cells as biocatalysts, thereby significantly improving the productivity of the acrylamide bioproduction process.