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2′-Hydroxyflavanone (2′OHFva), 3′OHFva, 4′OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3′OHFva and 4′OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4′OHFva (m/z 241) was used as a substrate, formation of 4′OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4′OHFvo by P450s. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes. [ABSTRACT FROM AUTHOR]

2′-, 3′-, and 4′-Methoxyflavones (MeFs) were incubated with nine forms of recombinant human cytochrome P450 (P450 or CYP) enzymes in the presence of an NADPH-generating system and the products formed were analyzed with LC-MS/MS methods. CYP1B1.1 and 1B1.3 were highly active in demethylating 4′MeF to form 4′-hydroxyflavone (rate of 5.0 nmol/min/nmol P450) and further to 3′,4′-dihydroxyflavone (rates of 2.1 and 0.66 nmol/min/nmol P450, respectively). 3′MeF was found to be oxidized by P450s to m/z 239 (M-14) products (presumably 3′-hydroxyflavone) and then to 3′,4′-dihydroxyflavone. P450s also catalyzed oxidation of 2′MeF to m/z 239 (M-14) and m/z 255 (M-14, M-14 + 16) products, presumably mono- and di-hydroxylated products, respectively. At least two types of ring oxidation products having m/z 269 fragments were formed, although at slower rates than the formation of mono- and di-hydroxylated products, on incubation of these MeFs with P450s; one type was products oxidized at the C-ring, having m/z 121 fragments, and the other one was the products oxidized at the A-ring (having m/z 137 fragments). Molecular docking analysis indicated the preference of interaction of O-methoxy moiety of methoxyflavones in the active site of CYP1A2. These results suggest that 2′-, 3′-, and 4′-methoxyflavones are principally demethylated by human P450s to form mono- and di-hydroxyflavones and that direct oxidation occurs in these MeFs to form mono-hydroxylated products, oxidized at the A- or B-ring of MeF. [ABSTRACT FROM AUTHOR]

The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2′-hydroxy-, 4′-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2′-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities. Molecular docking analysis showed that the C2′-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2′ and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes. [ABSTRACT FROM AUTHOR]

1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2′- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min−1 and 1.3 min−1, respectively. Other flavanone metabolites were 4′-, 3′- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min−1 that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min−1). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed. [ABSTRACT FROM AUTHOR]

For the precise estimation of the risk to human health caused by persistent organic pollutants (POPs), it is important to discuss enantiomer fraction value (EF value) because it is reported that behaviors such as stability and toxicity of enantiomers are quite different in human body. Among POPs, polychlorinated biphenyl (PCB) is known as one of the most persistent compounds in human breast milk samples. The main exposure source of PCB for human body is mostly from food especially in seafood. The contamination of fish and shellfish has been a serious problem for the Japanese, who consume a large amount of fish in their diet. PCBs have 19 congeners which are chlorine-substituted in 3- or 4- ortho positions are known to have enantiomers. In this study, we analyzed PCB 183 (2,2′,3,4,4′,5′,6-hepta CB) in human breast milk and fish samples enantioselectively and revealed the time trends of the EF value. Though EF value of PCB 183 in fish samples sustained close to racemate (EF = 0.5) from 1982 to 2012, that in breast milk increased over time. This fact indicates that (+)-PCB-183 has greater bioaccumulation potential than (-)-PCB-183 in human body; therefore, the toxicity of (+)-PCB-183 should be emphasized. [ABSTRACT FROM AUTHOR]

Atmospheric particles were collected in several cities in Japan (Sapporo, Sagamihara, Kanazawa, and Kitakyushu), Korea (Busan), and China (Beijing) using a high-volume air sampler equipped with a quartz fiber filter. The summer and winter samples were analyzed using gas chromatography-high-resolution mass spectrometry for Dechlorane Plus (DP). Decabromodiphenyl ether (BDE-209) was also analyzed for the samples from Kanazawa and Beijing. DP was detected in all samples. The mean total DP (ΣDP) concentration was highest (6.7 pg/m) and lowest (0.87 pg/m) in the winter samples from Sagamihara and Busan, respectively. The seasonal variation of DP concentrations varied by sampling site in this study. BDE-209 was detected in all the analyzed samples except for one of the Kanazawa winter samples. BDE-209 concentration was considerably higher in Beijing than in Kanazawa. Significant correlations were found between the concentrations of ΣDP and BDE-209 in the winter samples from Kanazawa and in both summer and winter samples from Beijing. This similarity in the atmospheric behavior of DP and BDE-209, especially in winter, is assumed to reflect a common end usage and release mechanism. [ABSTRACT FROM AUTHOR]

Polycyclicaromatic hydrocarbons (PAHs) and chlorinated PAHs (ClPAHs)are ubiquitous contaminants that bind to the aryl hydrocarbon receptor(AhR) and exhibit mutagenic potential. It is difficult to monitorhuman exposure levels to ClPAHs because the exposure routes are complicated,and environmental concentrations are not always correlated with thelevels of PAHs. Urinary PAH metabolites are useful biomarkers forevaluating PAH exposure, and ClPAH metabolites may therefore contributeto the estimation of ClPAH exposure. One of the most abundant ClPAHspresent in the environment is 1-chloropyrene (ClPyr), and urinaryClPyr metabolites have the potential to be good biomarkers to evaluatethe level of exposure to ClPAHs. Since the metabolic pathways involvingClPAHs are still undetermined, we investigated the effect of humancytochrome P450 enzymes on ClPyr and identified three oxidative metabolitesby liquid chromatography–tandem mass spectrometry and nuclearmagnetic resonance. We found that ClPyr was metabolized most efficientlyby the P450 1A1 enzyme, followed by the 1B1 and 1A2 enzymes. Similarto ClPyr, these metabolites were shown to have agonist activity forthe human AhR. We detected these metabolites when ClPyr reacted witha pooled human liver S9 fraction as well as in human urine samples.These results suggest that the metabolites may be used as biomarkersto evaluate the extent of exposure to ClPAHs. [ABSTRACT FROM AUTHOR]

Benzotriazole ultraviolet stabilizers (BUVSs) used in consumer products are raising concerns as new pollutants in the aquatic environment. We determined the agonistic activities of eight BUVSs and a chemically distinct UV absorber (4-methylbenzylidinecamphor) toward the human aryl hydrocarbon receptor (AhR) and thyroid hormone receptors alpha and beta. Although none of the BUVSs showed ligand activity against the thyroid hormone receptors, four of them (UV-P, UV-9, UV-326, and UV-090) showed significant AhR ligand activity. Their half-maximal effective concentrations (EC50) were 130 nM for UV-P, 460 nM for UV-9, and 5.1 μM for UV-090 (a value for UV-326 could not be determined). Of the numerous AhR ligands, it is well-known that those considered nontoxic are quickly metabolized by enzymes such as CYP1A1, which destroys their ability to function as ligands. Accordingly, we established a new yeast assay for simultaneous monitoring of both the strength of AhR ligand activity and ligand degradation by CYP1A1. We found the AhR ligand activities of the above four BUVSs to be stable in the presence of CYP1A1; therefore, they have the potential to accumulate and exert potent physiological effects in humans, analogous to polycyclic aromatic hydrocarbons and dioxins, which are known stable and toxic ligands. [ABSTRACT FROM AUTHOR]

The acetaldehyde associated with alcoholic beverages is an evident carcinogen for the esophagus. Genetic polymorphisms of the alcohol dehydrogenase 1 B ( ADH1 B) and aldehyde dehydrogenase 2 ( ALDH2) genes are associated with the risk of esophageal cancer. However, the exact mechanism via which these genetic polymorphisms affect esophageal carcinogenesis has not been elucidated. ADH1 B*2 is involved in overproduction of acetaldehyde due to increased ethanol metabolism into acetaldehyde, and ALDH2*2 is involved in accumulation of acetaldehyde due to the deficiency of acetaldehyde metabolism. Acetaldehyde can interact with DNA and form DNA adducts, resulting in DNA damage. N2-ethylidene-2′-deoxyguanosine ( N2-ethylidene-d G) is the most abundant DNA adduct derived from acetaldehyde. Therefore, we quantified N2-ethylidene-d G levels in blood samples from 66 Japanese alcoholic patients using liquid chromatography/electrospray tandem mass spectrometry, and investigated the relationship between N2-ethylidene-d G levels and ADH1 B and ALDH2 genotypes. The median N2-ethylidene-d G levels (25th percentile, 75th percentile) in patients with ADH1 B*1/* 1 plus ALDH2*1/* 1, ADH1 B*2 carrier plus ALDH2*1/* 1, ADH1 B*1/* 1 plus ALDH2*1/* 2, and ADH1 B*2 carrier plus ALDH2*1/* 2 were 2.14 (0.97, 2.37)/107 bases, 2.38 (1.18, 2.98)/107 bases, 5.38 (3.19, 6.52)/107 bases, and 21.04 (12.75, 34.80)/107 bases, respectively. In the ALDH2*1/* 2 group, N2-ethylidene-d G levels were significantly higher in ADH1 B*2 carriers than in the ADH1 B*1/* 1 group ( P < 0.01). N2-ethylidene-d G levels were significantly higher in the ALDH2*1/* 2 group than in the ALDH2*1/* 1 group, regardless of ADH1 B genotype ( ADH1 B*1/* 1, P < 0.05; ADH1 B*2 carriers, P < 0.01) N2-ethylidene-d G levels in blood DNA of the alcoholics was remarkably higher in individuals with a combination of the ADH1 B*2 and ALDH2*2 alleles. These results provide a new perspective on the carcinogenicity of the acetaldehyde associated with alcoholic beverages, from the aspect of DNA damage. [ABSTRACT FROM AUTHOR]

Abstract: We analyzed an acetaldehyde-derived DNA adduct, N 2-ethylidene-2′-deoxyguanosine (N 2-Eti-dG) in stomach DNA of aldehyde dehydrogenase (Aldh)-2-knockout mice that were fed with alcohol to determine effects of alcohol consumption and Aldh2 genotype on the level of DNA damage in stomach. Aldh2-active(+/+), heterozygote(+/−) and knockout(−/−) mice were fed 20% ethanol for 5 weeks, then the level of N 2-Eti-dG in stomach was determined by liquid chromatography tandem mass spectrometry. The average N 2-Eti-dG level in DNA from untreated mice was not significantly different among Aldh2 genotypes (2.0–3.1 adducts/107 bases), however, the average N 2 -Eti-dG level in DNA from ethanol-treated mice was 4.8±2.6 adducts/107 bases in Aldh2+/+ mice, 7.9±1.1 adducts/107 bases in Aldh2+/− mice, and 48.6±12.0 adducts/107 bases in Aldh2−/− mice, respectively. Our data clearly showed that alcohol drinking caused DNA damage in stomach, which was Aldh2 genotype-dependent in this experimental animal model. This result suggests that heavy-alcohol drinking and Aldh2 deficiency might be risk factors of stomach cancer. [Copyright &y& Elsevier]

A method was developed for the simultaneous determination of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish using ultra performance liquid chromatography with tandem mass spectrometry. Shellfish poisons in spiked samples were extracted with methanol and 90% methanol, and were hydrolyzed with 2.5 mol/L sodium hydroxide solution. Purification was done on an HLB solid-phase extraction column. This method was validated in accordance with the notification of Ministry of Health, Labour and Welfare of Japan. As a result of the validation study in nine kinds of shellfish, the trueness, repeatability and within-laboratory reproducibility were 79-101%, less than 12 and 16%, respectively. The trueness and precision met the target values of notification.

A method using dispersive and cartridge column solid-phase extraction (SPE) was developed for the simultaneous determination of veterinary drugs in livestock products by LC-MS/MS. The samples were extracted with 85% acetonitrile and cleaned up using dispersive and cartridge column SPE. Recoveries of 70 analytes fortified at the levels of 0.002 μg/g and 0.01 μg/g in cattle, swine and chicken muscles were examined. Among 70 analytes examined, 64 in cattle, 58 in swine and 49 in chicken satisfied the required Japanese validation guideline. The limit of quantitation values of the 70 analytes were 0.001 μg/g or 0.005 μg/g in three kinds of livestock products. This method is suitable to analyze 40 analytes in three kinds of livestock products at levels under the MRL.

Human body might be exposed to acetaldehyde from smoking or occupational environment, which is known to be associated with cancer through the formation of DNA adducts, in particular, N2-ethylidene-2'- deoxyguanosine (N2-ethylidene-dG). Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that contribute to the detoxification of acetaldehyde in human body. In this study, wild type (Aldh2+/+) and Aldh2KO (Aldh2-/-) mice were exposed to the air containing 0, 125, 500 ppm acetaldehyde for 2 weeks. After inhalation, levels of N2- ethylidene-dG in the chromosomal DNA were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). N2-ethylidene-dG levels in livers of Aldh2-/- mice were always lower than those of Aldh2+/+ mice, suggesting that Aldh2 deficiency might cause the induction of acetaldehyde metabolizing enzymes in the liver such as P450s. The differences between Aldh2-/- and Aldh2+/+ mice were greater in the order of nasal epithelium > lung > dorsal skin, suggesting that nasal epithelium and lung are the major target sites for acetaldehyde. Acetaldehyde inhalation may cause a high risk in nasal epithelium and lung cancers for individuals with inactive ALDH2.

To conduct proficiency testing for the analysis of pesticide residues in processed foods, fortified samples of retort curry and pancake were examined. In the case of retort curry, heating and mixing were necessary at the time of preparation to provide a homogenous analytical sample. A mixture of 4 carbamates and 11 organophosphorus pesticides was spiked and 14 of them showed consistent results in the samples. In the case of pancake, 10 kinds of pesticides were added to the pastry. The prepared pastry was them cooked. The relative concentrations of most of the pesticides in the pancake were not affected and all the pesticides showed consistent results in the samples. These results showed that the two tested samples were suitable for proficiency testing.

A method using liquid chromatograph coupled with tandem mass spectrometer (LC/MS/MS) was developed for the determination of melamine in processed food. After extraction with 50% acetonitrile and clean-up with PSA and SCX, the quantification limit of melamine in processed food was 0.5 microg/g. The recoveries and relative standard deviations were 113 to 115% and less than 5%, respectively. Therefore, we considered that the developed method offers high precision and sensitivity for the determination of melamine in processed food. When the method was applied to six suspected products, 0.8 to 37 microg/g of melamine was detected from four milk-rich products.