Matt J. Hengel

Carcinogenic 4(5)-Methylimidazole Found in Beverages, Sauces, and Caramel Colors: Chemical Properties, Analysis, and Biological Activities

Since the National Toxicology Program (NTP) identified 4(5)-methylimidazole [4(5)-MI] as a cancer causing chemical in 2007 and the State of California added it to the Proposition 65 list of compounds as a carcinogen on January 7, 2011, many researchers and regulatory agencies have become focused on the presence of 4(5)-MI in foods and beverages. 4(5)-MI has been known to form in the Maillard reaction system consisting of a sugar and ammonia-a typical caramel-color preparation method for beverages. 4(5)-MI is identified in various beverages and sauces, which are colored with caramel, as well as in caramel color itself. Analysis of 4(5)-MI is extremely difficult due to its high water solubility, but the analytical method for 4(5)-MI has progressed from conventional paper chromatography, gas chromatography, and gas chromatography-mass spectrometry to the most advanced high-performance liquid chromatography-mass spectrometry. Various studies indicate that caramel colors and carbonated beverages contain 4(5)-MI in levels ranging from 0 to around 1000 ppm and from 0 to about 500 ppm, respectively. Reports of the toxicity of 4(5)-MI at relatively high levels suggest that it may cause some adverse effects on human consumers.

Formation of Acrylamide from Lipids

Heating amino acids with dietary oils or animal fats at elevated temperatures produced various amounts of acrylamide. The amount of acrylamide formation corresponded to the degree of unsaturation of the oils and animal fats. The decreasing order of acrylamide formation from dietary oils or animal fats with asparagine was sardine oil (642 microg/g asparagine) > cod liver oil (435.4 microg/g) > soybean oil (135.8 microg/g) > corn oil (80.7 microg/g) > olive oil (73.6 microg/g) > canola oil (70.7 microg/g) > corn oil (62.1 microg/g) > beef fat (59.3 microg/g) > lard (36.0 microg/g). Three-carbon unit compounds such as acrylic acid and acrolein, which are formed from lipids by oxidation also produced acrylamide by heat treatment with amino acids, in particular with asparagine. The results of the present study suggest that acrylamide forms in asparagine-rich foods during deep fat frying in the absence reducing sugars.

Evidence for Trichloroethylene Bioactivation and Adduct Formation in the Rat Epididymis and Efferent Ducts

Abstract Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome P450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate β-lyase (β-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of β-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of β-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome P450-mediated pathway. Western blot analysis detected the presence of cytochrome P450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome P450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.

Determination of Toxic α-Dicarbonyl Compounds, Glyoxal, Methylglyoxal, and Diacetyl, Released to the Headspace of Lipid Commodities upon Heat Treatment

Toxic α-dicarbonyl compounds, glyoxal, 2-methylglyoxal, and diacetyl, released from the headspace from butter, margarine, safflower oil, beef fat, and cheese heated at 100 and 200 °C were analyzed by gas chromatography as quinoxaline derivatives. Total amounts of α-dicarbonyl compounds ranged from 40.5 ng/g (butter) to 331.2 ng/g (beef fat) at 100 °C and from 302.4 ng/g (safflower oil) to 4521.5 ng/g (margarine) at 200 °C. The total amount of α-dicarbonyl compounds increased approximately 55- and 15-fold in the headspace of heated butter and margarine, respectively, when the temperature was increased from 100 to 200 °C. However, only slight differences associated with temperature variation were observed in the cases of safflower oil and beef fat (1.3- and 1.1-fold, respectively). Diacetyl was found in the highest amounts among all samples, ranging from 13.9 ± 0.3 ng/g (butter) to 2835.7 ng/g (cheese) at 100 °C and from 112.5 ± 102 ng/g (safflower oil) to 2274.5 ± 442.6 ng/g (margarine) at 200 °C, followed by methylglyoxal, ranging from 13.0 ± 0.5 to 112.7 ± 10.1 ng/g (cheese) at 100 °C and from 34.7 ± 5.0 ng/g (safflower oil) to 1790 ± 372.3 ng/g (margarine) at 200 °C. Much less glyoxal formed, in amounts ranging from 13.6 ± 0.7 ng/g (butter) to 53.4 ± 11.2 ng/g (beef fat) at both temperatures. The amounts of α-dicarbonyl compounds released into the vapor phase from lipid commodities during heating were satisfactorily analyzed.

Formation of 4(5)-methylimidazole and its precursors, α-dicarbonyl compounds, in Maillard model systems.

Glyoxal, methylglyoxal, and diacetyl formed from sucrose alone and from a D-glucose/ammonia Maillard model system were analyzed by gas chromatography. They are known as precursors of 4(5)-methylimidazole (MI). Glyoxal and methylglyoxal formed more in acidic conditions than in basic conditions, whereas diacetyl formed the most at the highest basic condition of pH 12. Glyoxal formation from sucrose ranged from 0.33 to 32.90 μg/g under four different time and temperature conditions. Amounts of glyoxal, methylglyoxal, and diacetyl formed in Maillard model systems ranged from 2.98 to 46.12 μg/mL, from 8.27 to 156.61 μg/mL, and from 14.94 to 1588.45 μg/mL, respectively. 4(5)-MI formation in the same model systems ranged from 28.56 to 1269.71 μg/mL. Addition of sodium sulfite reduced formation of these chemicals significantly. Total α-dicarbonyl compounds in 12 commercial soft drinks ranged from 5.75 to 50.72 μg/mL. 4(5)-MI was found in levels ranging from 1.76 to 28.11 ng/mL in 10 commercial soft drinks.

Analysis of pesticides in dried hops by liquid chromatography-tandem mass spectrometry.

An analytical method was developed for the determination of eleven agrochemicals [abamectin (as B1a), bifenazate, bifenthrin, carfentrazone-ethyl, cymoxanil, hexythiazox, imidacloprid, mefenoxam, pymetrozine, quinoxyfen, and trifloxystrobin] in dried hops. The method utilized polymeric and NH2 solid phase extraction (SPE) column cleanups and liquid chromatography with mass spectrometry (LC-MS/MS). Method validation and concurrent recoveries from untreated dried hops ranged from 71 to 126% for all compounds over three levels of fortification (0.10, 1.0, and 10.0 ppm). Commercially grown hop samples collected from several field sites had detectable residues of bifenazate, bifenthrin, hexythiazox, and quinoxyfen. The control sample used was free of contamination below the 0.050 ppm level for all agrochemicals of interest. The limit of quantitation and limit of detection for all compounds were 0.10 and 0.050 ppm, respectively.

Effect of Acinetobacter sp on Metalaxyl Degradation and Metabolite Profile of Potato Seedlings (Solanum tuberosum L.) Alpha Variety

One of the most serious diseases in potato cultivars is caused by the pathogen Phytophthora infestans, which affects leaves, stems and tubers. Metalaxyl is a fungicide that protects potato plants from Phytophthora infestans. In Mexico, farmers apply metalaxyl 35 times during the cycle of potato production and the last application is typically 15 days before harvest. There are no records related to the presence of metalaxyl in potato tubers in Mexico. In the present study, we evaluated the effect of Acinetobacter sp on metalaxyl degradation in potato seedlings. The effect of bacteria and metalaxyl on the growth of potato seedlings was also evaluated. A metabolite profile analysis was conducted to determine potential molecular biomarkers produced by potato seedlings in the presence of Acinetobacter sp and metalaxyl. Metalaxyl did not affect the growth of potato seedlings. However, Acinetobacter sp strongly affected the growth of inoculated seedlings, as confirmed by plant length and plant fresh weights which were lower in inoculated potato seedlings (40% and 27%, respectively) compared to the controls. Acinetobacter sp also affected root formation. Inoculated potato seedlings showed a decrease in root formation compared to the controls. LC-MS/MS analysis of metalaxyl residues in potato seedlings suggests that Acinetobacter sp did not degrade metalaxyl. GC–TOF–MS platform was used in metabolic profiling studies. Statistical data analysis and metabolic pathway analysis allowed suggesting the alteration of metabolic pathways by both Acinetobacter sp infection and metalaxyl treatment. Several hundred metabolites were detected, 137 metabolites were identified and 15 metabolic markers were suggested based on statistical change significance found with PLS-DA analysis. These results are important for better understanding the interactions of putative endophytic bacteria and pesticides on plants and their possible effects on plant metabolism.

Development and Validation of a Standardized Method for the Determination of Morpholine Residues in Fruit Commodities by Liquid Chromatography–Mass Spectrometry

An analytical method was developed for the determination of morpholine on apples and citrus. The method utilized acidified methanol extraction, centrifugation, and determination by hydrophilic interaction liquid chromatography with electrospray ionization and tandem mass spectrometry (HILIC-ESI-MS/MS). Validation of the method occurred at the Pacific Agricultural Laboratory (PAL, Portland, OR, USA) and the Trace Analytical Laboratory (TAL, UC Davis, CA, USA). Method validation recoveries from control apple, orange, lemon, and grapefruit samples ranged from 84 to 120% over three levels of fortification (0.01, 0.04, and 0.2 μg/g). The limit of quantitation (LOQ) for all commodities was 0.01 μg/g, and the calculated method detection limit (MDL) ranged from 0.0010 to 0.0040 μg/g.

Comparison of Direct and Indirect Photolysis in Imazosulfuron Photodegradation

Imazosulfuron, a sulfonylurea herbicide used in rice cultivation, has been shown to undergo photodegradation in water, but neither the photochemical mechanism nor the role of indirect photolysis is known. The purpose of this study was to investigate the underlying processes that operate on imazosulfuron during aqueous photodegradation. Our data indicate that in the presence of oxygen, most photochemical degradation proceeds through a direct singlet-excited state pathway, whereas triplet-excited state imazosulfuron enhanced decay rates under low dissolved oxygen conditions. Oxidation by hydroxyl radical and singlet oxygen were not significant. At dissolved organic matter (DOM) concentrations representative of rice field conditions, fulvic acid solutions exhibited faster degradation than rice field water containing both humic and fulvic acid fractions. Both enhancement, via reaction with triplet-state DOM, and inhibition, via competition for photons, of degradation was observed in DOM solutions.

Isolation of a Potent Naturally Occurring Hallucinogen, Salvinorin A, from Salvia divinorum and Investigation of Its Photo-degradation

Salvinorin A, which is present in the natural plant Salvia divinorum, is a unique non-nitrogenous compound with hallucinogenic activity. When salvinorin A isolated from leaves of Salvia divinorum was irradiated with 300 nm UV light in ethyl acetate, it degraded from 100 μg/mL to 2.84 ± 0.05 μg/mL in 30 min. The calculated average rate constant k of this degradation was 0.12/min and the half-life was 5.7 min. When authentic salvinorin A was irradiated by UV light in an organic solution or an aqueous solution, it degraded over 90% within 40 min, whereas when it was irradiated by natural sunlight, it took 8 h to degrade 50% both in an organic and an aqueous solution. Three photodegradation products were tentatively identified by gas chromatography/mass spectrometry. Their structures were similar to that of salvinorin A, suggesting that they are also candidate non-nitrogenous hallucinogens.