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4-methylimidazole (4-MI), a derivative of imidazole, is a widely used component in caramel-colored food products such as soy sauce, beer and other soft drinks. The present study is aimed to investigate the effects of 4-MI on the male reproduction. The results revealed that 8 weeks of 4-MI exposure did not significantly alter the body weight and testicular weight of male mice. However, testicular morphology and computer-assisted sperm analysis showed that exposed to 4-MI caused irregular arrangement of spermatogenic cells in the testes and weakened sperm motility. Consistently, we observed the decreased fertilization ability in vivo of 4-MI-treated male mice. We further demonstrated that 4-MI disrupted the blood-testis barrier (BTB) integrity by decreasing the protein expression of BTB-related junction with permeability assay and western blot. In addition, the apoptosis of Sertoli cells (TM4) occurred in 4-MI treated mice, which might be caused by the generation of oxidative stress. Collectively, our findings document that 4-MI exposure damages the sperm mobility via disruption of BTB integrity.

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4(5)-methylimidazole (4-MeI) is a potential carcinogen widely used in food colours. EU regulations specify a maximum allowable concentration of 200 ppm for 4-MeI in caramel colours. This study reports an electrochemical determination technique for 4-MeI in caramel colours for the first time. The effect of pH and interference from air were studied to optimize the detection conditions on a glassy carbon electrode in aqueous alkaline solutions using square wave voltammetry (SWV) technique. The concentration of 4-MeI was quantitatively measured down to 10 µM (∼0.8 ppm). Traditional methods such as HPLC, GC, spectrometry and immunoassays involve either expensive instrumentation and reagents or time consuming preparation and detection processes. This study demonstrates the possibility of rapid and simple electrochemical determination of (4-MeI) in food colours with minimum workup using a portable potentiostat.

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4-Methylimidazole (4-MEI), as a Maillard reaction product, often occurs in heat-processed food. Due to its widespread occurrence and strong carcinogenicity in food and beverages, 4-MEI has received attention from regulatory organizations and consumers. Some studies have reported the occurrence and exposure of 4-MEI in food, but few studies have involved traditional tea beverages, which is related to the limited analytical methods currently being influenced by complex tea matrices. For this issue, this study presents a simple, reliable, and highly sensitive analytical method for the determination of 4-MEI in tea using liquid chromatography-high resolution mass spectrometry. By means of this method, a total of 570 tea samples from typical tea-producing regions in China were monitored for contamination of 4-MEI. The results showed that the average 4-MEI level (136 µg/kg) in oolong tea was significantly higher than that in other types of tea samples. Based on contamination levels and tea consumption data in China, the daily intake doses (0.04-1.16 µg/day) of 4-MEI among tea consumers were obtained. As a result, the health risk of Chinese tea consumers consuming 4-MEI alone through tea consumption is relatively low, but the overall intake level of 4-MEI in other foods cannot be ignored.

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Compromised maternal health leading to maternal seizures can have adverse effects on the healthy development of offspring. This may be the result of inflammation, hypoxia-ischemia, and altered GABA signaling. The current study examined cortical tissue from F2b (2nd litter of the 2nd generation) postnatal day 4 (PND4) offspring of female Harlan SD rats chronically exposed to the seizuregenic compound, 4-Methylimidazole (0, 750, or 2500 ppm 4-MeI). Maternal seizures were evident only at 2500 ppm 4-MeI. GABA related gene expression as examined by qRT-PCR and whole genome microarray showed no indication of disrupted GABA or glutamatergic signaling. Canonical pathway hierarchical clustering and multi-omics combinatory genomic (CNet) plots of differentially expressed genes (DEG) showed alterations in genes associated with regulatory processes of cell development including neuronal differentiation and synaptogenesis. Functional enrichment analysis showed a similarity of cellular processes across the two exposure groups however, the genes comprising each cluster were primarily unique rather than shared and often showed different directionality. A dose-related induction of cytokine signaling was indicated however, pathways associated with individual cytokine signaling were not elevated, suggesting an alternative involvement of cytokine signaling. Pathways related to growth process and cell signaling showed a negative activation supporting an interpretation of disruption or delay in developmental processes at the 2500 ppm 4-MeI exposure level with maternal seizures. Thus, while GABA signaling was not altered as has been observed with maternal seizures, the pattern of DEG suggested a potential for alteration in neuronal network formation.

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The inhibitory effects of liquiritigenin, liquiritin and glycyrrhizic acid against the hazards during the preparation of thermal reaction beef flavoring were investigated using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Liquiritigenin(1.5 mM) inhibited Nε-carboxymethyl-L-lysine and Nε-carboxyethyl-L-lysine by up to 38.69 % and 61.27 %, respectively; 1.5 mM liquiritin inhibited 4-methylimidazole by up to 48.28 %; and 1.5 mM liquiritigenin and 1.0 mM liquiritin inhibited hydroxymethylfurfural by up to 61.20 % and 59.31 %, respectively. The results of the model system showed that the inhibitory effect of the 3 inhibitors could be extended to other thermal reaction flavoring systems. The 3 inhibitors can effectively block key intermediates in beef flavoring, and liquiritigenin can inhibit up to 22.97 % of glyoxal and 22.89 % of methylglyoxal. In addition, liquiritigenin and liquiritin can directly eliminate up to 25.87 % and 21.01 % of methylglyoxal by addition and other means. Free radicals in the simultaneous formation model system were measured using electron spin resonance (ESR), and the results showed that liquiritigenin, liquiritin and glycyrrhizic acid could scavenge free radicals in the system in a dose-dependent manner, with scavenging rates of up to 44.88-57.09 %. Therefore, the inhibitory effects of the 3 inhibitors can be attributed to the intermediate blocking and free radical scavenging pathways.

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A qualitative and quantitative method for detecting free and protein-bound advanced glycation end products (AGEs) and 4-methylimidazole (4-MI) was established using isotope dilution-HPLC-MS/MS, and successfully applied in cookies and model systems. The effects of different temperatures (160-220 °C) on the formation of free and protein-bound AGEs and 4-MI in cookies were discussed, and the possible model systems (Maillard reaction pathway 1 using wheat gluten protein + glucose + sucrose; direct addition pathway 1 using wheat gluten protein + CML/CEL/4-MI) of protein-bound AGEs and 4-MI were verified. The results showed that the contents of protein-bound CML, CEL, and 4-MI were higher than free content with a tendency of increasing first and subsequently decreasing with temperature, reaching a maximum at 200 °C in cookies. In the model systems, the levels of protein-bound CML, CEL, and 4-MI are higher than those of free CML, CEL, and 4-MI. The protein-bound CML, CEL, and 4-MI accounted for 90.73, 87.64, and 97.56% of the total amount in the model system 1, while accounting for 68.19, 59.00, and 50.96% in the model system 2, respectively. In comparison, protein-bound CML, CEL, and 4-MI could be easily generated directly by Maillard reaction.

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4-methylimidazole (4-MEI) is widely used industrially. This carcinogenic component has been reported in some types of food. It is usually produced by the caramelization process in food, drinks and caramel coloring. The possible mechanism for the formation of this compound in food is the Maillard reaction. In order to estimate the amount of substance 4-MEI in food, a systematic study was conducted. The selected keywords were 4-methylimidazole, 4-MEI, beverage, drink, meat, milk, and coffee. 144 articles were obtained from the initial search. The articles were evaluated and finally, the data of 15 manuscripts were extracted. Based on the data extracted from selected articles, the highest amount is reported in caramel color, coffee, and cola drinks. In 70% of the selected studies, the analytical method was based on liquid chromatography. In this method, there is no need for derivatization. SPE columns were used to extract samples in most manuscripts. According to per capita consumption, the most exposure to 4-MEI is through coffee. In high risk food products, regular monitoring with analytical methods with high sensitivity is recommended. Furthermore, most of the selected studies were about the validation method, so few samples were selected. It is recommended to design more studies with a high sample size to accurately evaluate this carcinogenic compound in food.

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BACKGROUND: 4-Methylimidazole (4-MeI) or 4-methyl-1H-imidazole, a slightly yellowish solid with molecular formula C4H6N2, is a heterocyclic compound which supposedly does not exist as a natural product and is formed when carbohydrates are heating with ammonium compounds. This compound is used in pharmaceuticals, agriculture and photography chemicals, dyes and pigments, and rubber manufacturing. In the present study, a simple and efficient sample preparation method designated gas flow headspace liquid phase microextraction (GF-HS-SDME) was employed for the extraction and preconcentration of 4-methylimidazole (4-MeI) from food and beverage samples, before its determination by gas chromatography-mass spectrometry. RESULT: To investigate the optimal conditions for the extraction process in GF-HS-SDME method, factors affecting extraction, including selection of extraction solvent, vial volume, extraction solvent ratio, position of extracting solvent, drop volume, sample volume, stirring speed, temperature, extraction time, sample pH, ionic strength of the sample solution and gas flow rate were optimized by utilizing both one-variable-at-a-time method and Plackett-Burman design. The investigation of protocol was carried out by using a standard solution containing 100.0 µg L-1 of 4-MeI in deionized water. CONCLUSION: In this study, a simple and green analytical method based on GF-HS-SDME was proposed for the extraction and preconcentration of 4-MeI from foodstuffs, followed by GC-MS determination. The main advantage of this method is its high preconcentration factor and fastness due to the application of an inert gas stream during microextraction.

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Individuals of all ages, including children and teenagers, consume 4-methylimidazole (4-MI) in their food. 4-MI is a caramel-colored waste product that has previously been linked to human carcinogenesis and has shown possible signs of reproductive toxicity. This study aimed to determine whether 4-MI is harmful to oocytes during meiosis and fertilization. Female mice were intragastrically administered 0, 50, or 100 mg/kg body weight of 4-MI daily for 10 days. We found that 4-MI affects the quality of oocytes by affecting their meiotic ability and fertility potential. Specifically, 4-MI rendered the meiotic spindles and chromosomes less stable, which halted oocyte maturation and resulted in aneuploidy. 4-MI also slowed the decrease in the levels of cortical granules and their component ovastacin; consequently, sperms could not be bound and fertilization could not occur. We also found that mitochondrial dysfunction was associated with oocytes deterioration. This led to reactive oxygen species accumulation and cell death. Altogether, our findings reveal that the poor condition of oocytes subjected to 4-MI is primarily attributable to mitochondrial malfunction and redox alterations.

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Herein, a facile and fast surface-enhanced Raman scattering (SERS) method with ratiometric strategy was developed for detection of 4-methylimidazole (4-MI). Via a chemical derivatization reaction with 3-amino-5-mercapto-1,2,4-triazole (AMTA) diazonium salts, 4-MI could be converted to SERS-sensitive species. The SERS intensity ratio between the peaks at 1243 cm-1 and 1110 cm-1 (I1243/I1110) was used for the quantification of 4-MI. In addition, the method sensitivity was further improved by the aggregation of beta-cyclodextrin-modified Ag nanoparticles (beta-CD-AgNPs). Under the optimal conditions, the limit of detection (LOD) and the limit of quantification (LOQ) for 4-MI were 1.7 nM (S/N = 3) and 5.7 nM (S/N = 10), respectively. The relative standard deviation (RSD) for 0.5 µM 4-MI was 8.2% (n = 20). This method was successfully used for the determination of 4-MI in cola samples with recoveries ranging from 92% to 106%. The present method is convenient, sensitive, selective, reliable and may have a promising application in determination of the compounds with an imidazole ring containing active hydrogen atoms.

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The National Toxicology Program (NTP) reported that chronic exposure to varying dietary concentrations of 4-methylimidazole (4-MeI) increased lung tumors in female and male mice [1]. In this study, mice (male and female B6C3F1 mice) were either administered 4-MeI by oral gavage (0, 50, 100, 200, or 300 mg/kg/day) for 2 days or exposed for 5 and 28 days to 4-MeI in the diet (0, 150, 300, 1250, or 2500 ppm) and whole transcriptome (RNA-Sequencing) data from 4-MeI-exposed B6C3F1 mice to determine whether changes occurred in the target (lung) and nontarget (liver) tissues. This analysis was conducted to provide information with which to evaluate biological processes affected by exposure to 4-MeI, with a focus on identifying key events that could be used to propose a plausible mode of action (MoA) for mouse lung tumors [2].

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The National Toxicology Program (NTP) reported that chronic dietary exposure to 4-methylimidazole (4-MeI) increased the incidence of lung adenomas/carcinomas beyond the normally high spontaneous rate in B6C3F1 mice. To examine plausible modes of action (MoAs) for mouse lung tumors (MLTs) upon exposure to high levels of 4-MeI, and their relevance in assessing human risk, a systematic approach was used to identify and evaluate mechanistic data (in vitro and in vivo) in the primary and secondary literature, along with high-throughput screening assay data. Study quality, relevance, and activity of mechanistic data identified across the evidence-base were organized according to key characteristics of carcinogens (KCCs) to identify potential key events in known or novel MLT MoAs. Integration of these evidence streams provided confirmation that 4-MeI lacks genotoxic and cytotoxic activity with some evidence to support a lack of mitogenic activity. Further evaluation of contextual and chemical-specific characteristics of 4-MeI was consequently undertaken. Due to lack of genotoxicity, along with transcriptomic and histopathological lung changes up to 28 and 90 days of exposure, the collective evidence suggests MLTs observed following exposure to high levels of 4-MeI develop at a late stage in the mouse chronic bioassay, albeit the exact MoA remains unclear.

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4-Methylimidazole (4-MeI) is a byproduct formed during the cooking of foods containing carbohydrates and amino acids, including the production of flavors and coloring substances, e.g., class III and IV caramel colors, used in many food products with extensive human exposure. Two-year rodent bioassays via oral exposure conducted by the National Toxicology Program reported evidence of carcinogenicity only in B6C3F1 mice (increased alveolar/bronchial neoplasms). In 2011, the International Agency for Research on Cancer classified 4-MeI as Group 2B, "possibly carcinogenic to humans". An expert panel was commissioned to assess the genotoxic potential of 4-MeI and the plausibility of a genotoxic mode of action in the formation of lung tumors in mice when exposed to high doses of 4-MeI. The panel defined and used a weight-of-evidence (WOE) approach that included thorough evaluation of studies assessing the genotoxic potential of 4-MeI. The panelists categorized each study, consisting of study weight, degree of technical performance, study reliability, and contribution to the overall WOE. Based on the reviewed studies' weighted contribution, the panel unanimously concluded that the WOE supports no clear evidence of in vivo genotoxicity of 4-MeI and no association for a genotoxic mode of action in the formation of mouse lung tumors.

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4-Methylimidazole (4MEI) is a contaminant in food and consumer products. Pulmonary toxicity and carcinogenicity following chronic dietary exposures to 4MEI is a regulatory concern based on previous rodent studies. This study examined acute pulmonary toxicity in B6C3F1 mice from 6 h to 5 days after oral gavage with a single dose of 150 mg/kg 4MEI, a double dose delivered 6 h apart, or vehicle controls. Oral gavage of 150 mg/kg naphthalene, a prototypical Club cell toxicant, was used as a positive control. Intrapulmonary conducting airway cytotoxicity was assessed in fixed-pressure inflated lungs using qualitative histopathology scoring, quantitative morphometric measurement of vacuolated and exfoliating epithelial cells, and immunohistochemistry. 4MEI treatment did not change markers of cytotoxicity including the mass of vacuolated epithelium, the thickness of the epithelium, or the distributions of epithelial proteins: secretoglobin 1A1, proliferating cell nuclear antigen, calcitonin gene-related peptide, and myeloperoxidase. 4MEI and vehicle controls caused slight cytotoxicity with rare vacuolization of the epithelium relative to the severe bronchiolar epithelial cell toxicity found in the naphthalene exposed mice at terminal bronchioles, intrapulmonary airways, or airway bifurcations. In summary, 4MEI caused minimal airway epithelial toxicity without characteristic Club Cell toxicity when compared to naphthalene, a canonical Club Cell toxicant.

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The general population, including children and adolescents, is exposed to 4-methylimidazole (4-MI) in the diet. 4-MI is a by-product of caramel color manufacturing. It has been previously classified as a possible human carcinogen and displays potential reproductive toxicity. A follow up assessment of reproductive toxicity was conducted in rats utilizing the reproductive assessment by continuous breeding paradigm, in which multiple generations were exposed to 4-MI in diet at 750, 2500, and 5000 ppm. 4-MI exposure was associated with delays in preputial separation and vaginal opening, impairment in reproductive performance, and concomitant histopathological findings in the prostate, testis, and epididymis at 2500 and 5000 ppm. The Lowest Observed Adverse Effect Level for reproductive (based on prostate atrophy) and developmental toxicity (based on delays in preputial separation and vaginal opening) was 750 ppm, equivalent to approximately 50-60 mg/kg bw/day.

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This study investigated the effect of chemical substances, such as Mg2+ (as magnesium sulphate), Zn2+ (as zinc sulphate), Fe2+ (as iron sulphate), ascorbic acid, and citric acid, on the formation of 4-methylimidazole (4-MI) and colour stability of caramel colour. The 4-MI concentration in the caramel colour without chemical substances was 963.10 µg/g, and this decreased the most (67.7%) with the addition of 0.1 molar ratio of citric acid. Colour stability was evaluated by measuring the total colour change (ΔE) after storage, and heating in pH 2, 4, and 7 solutions and 50% solution in ethyl alcohol. Among the chemical substances added to reduce 4-MI in the caramel colour, Mg2+ (0.1 molar ratio) and ascorbic acid improved the colour stability more than the others.

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4-Methylimidazole (4-MeI) is a nitrogen-containing heterocyclic compound that is used in the manufacture of chemicals, dyes and pharmaceuticals and may be found in a variety of foods following formation during heating. The purpose of this study was to use two different in silico programs, CASE Ultra and Toxtree, to investigate potential structure-activity relationships in 4-MeI and its metabolites for mutagenicity and carcinogenicity, and combine that information with the available literature to draw conclusions regarding the strength of the predictions observed. Neither CASE Ultra nor Toxtree identified any structural alerts that were associated with mutagenic activity. Data for 4-MeI from a single study were used in the development of the CASE Ultra mouse and rat carcinogenicity models, but no additional similar structures were identified in the carcinogenicity model training set. One metabolite, 5-methylhydantoin, was predicted to be positive in the CASE Ultra carcinogenicity male and female mouse models; positive predictivity percentages of 60.9% and 73.7%, respectively. However, low structural similarity between 5-methylhydantoin and the compounds identified in the training set (<25%) decreases confidence in the positive prediction. Three metabolites were predicted to be positive in the CASE Ultra mouse micronucleus model, but again suffered from low structural similarity. Both limited structural similarity and inconsistent responses among the other clastogenicity models suggest that additional structurally similar compounds are needed to assess the predictive capacity of these alerts for biological activity of these compounds.

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A sensitive method using ion-pair extraction was developed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for measurement of 4-methylimidazole (4-MI) in NMRI mice plasma and cerebrospinal fluid (CSF). Detection was done by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The validation method was applied to quantification of 4-MI in plasma and CSF samples using oral doses of 100, 200, and 300 mg/kg in NMRI mice. The efficiency of the method was evaluated in terms of linearity (R 2> 0.99), recovery (98-107%, 3 levels) and precision (8-10%, 3 levels, n = 6). Limit of detection (LOD) and limit of quantification (LOQ) were 25 ng/mL and 50 ng/mL, respectively. The results obtained showed that the exposure to oral doses of 4-MI in mice makes different concentrations in plasma and CSF and causes significant changes in mice. This study was the first report for determination of 4-MI in plasma and CSF samples in mice. Our results suggest that LC-MS/MS-based on ion-pair extraction is a robust method with high detection ability for measurement of 4-MI in plasma and CSF samples. Therefore, the developed method can be useful for evaluation and monitoring of imidazole derivatives in biological samples.

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4(5)-Methylimidazole (4(5)-MI) is a potential carcinogen with low molecule weight, highly polarity, and weak basicity. The traditional way to extract and clean-up 4(5)-MI in soy sauce using solid phase extraction is tedious and time consuming. Here we proposed a method for the determination of 4(5)-MI in soy sauce by combining a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction with liquid chromatography-mass spectrometry analysis. The impacts of solution pH, water addition, and cleanup procedure on 4(5)-MI extraction efficiency were studied. An optimized sample preparation approach involved a single step liquid-liquid extraction between acetonitrile and soy sauce under alkaline conditions, followed by primary and secondary amine clean-up. The analytical method was validated with soy sauce at three spiking levels (10, 50, 500 ng/g). The method recovery (96.2-107%) and intra-day/inter-day precision (4.1-8.4%/6.9-11.7%) were satisfactory. The method quantification limit was 10 ng/g. The developed method was successfully applied for the determination of 4(5)-MI in fourteen commercial soy sauces from local markets. The results obtained in this work suggests that the method is suitable for the analysis of 4(5)-MI at low concentrations in high-salting and protein-containing soy sauce matrix.

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2-Methylimidazole, 4-methylimidazole and 5-hydroxymethylfurfural are harmful by-products potentially formed via Maillard reaction in fermented soy sauce. The present study proposed a new method based on "quick, easy, cheap, effective, rugged, and safe" purification and ultra high performance liquid chromatography with tandem mass spectrometry for the simultaneous analysis of 2-methylimidazole, 4-methylimidazole and 5-hydroxymethylfurfural in fermented soy sauce. The sample was dissolved in water after addition of internal standard 4-methylimidazole-d6 and extracted with acetonitrile. After dehydration, it was centrifuged and the supernatant was subsequently purified using two sorbents namely primary-secondary amine and multi-walled carbon nanotube. Three target analytes were separated by gradient elution and determined under multiple reactions monitoring mode. The limit of detection, matrix effect, recovery and precision of the developed method were investigated. Results found that three target analytes displayed excellent linearity in concentration range of 1-250 µg/L. Limit of detection was in the range of 0.3-1 µg/kg for three target analytes. The mean recoveries for fermented soy sauce samples at three spiked concentrations were in the range of 91.2-112.5%, and the intra- and interday precision were in the ranges of 3.6-9.2 and 7.1-10.8%, respectively. This validated method was successfully applied to determine 2-methylimidazole, 4-methylimidazole and 5-hydroxymethylfurfural concentrations in fermented soy sauce.

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