Blue Chitin Research Articles

Copper Phthalocyanine Sulfonate-mediated Oxidative Degradation of 3-Hydroxyamino-1-methyl-5H-pyrido [4,3-b]indole [Trp-P-2(NHOH)], a Direct-acting Mutagen Derived from Trp-P-2 by Metabolic Activation

Blue rayon and blue chitin are potential absorbents of heterocyclic amines. However, in our preliminary research, no recovery (0%) of an activated form of Trp-P-2, 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2 (NHOH)] with blue rayon extraction was observed. We investigated whether copper phthalocyanine sulfonate on blue rayon may react with Trp-P-2(NHOH) and accelerate the degradation of Trp-P-2(NHOH) in concert with oxygen and photons. Both the disulfonate and tetrasulfonate of copper phthalocyanine inhibited the mutagenicity of Trp-P-2(NHOH). Copper phthalocyanine disulfonate (CuCYB) accelerated the disappearance of Trp-P-2(NHOH). The degradation of Trp-P-2(NHOH) with CuCYB was attenuated in the presence of glutathione, under dark conditions, and with helium bubbling. Therefore, the reaction mechanisms may involve the interaction of molecular oxygen with CuCYB, which accelerate the oxidative degradation of Trp-P-2(NHOH). Photodynamic activation of molecular oxygen might contribute, in a part, to the oxidative degradation of Trp-P-2(NHOH).

On-line capillary based quantitative analysis of a heterocyclic amine in human urine

A high through-put miniaturised separation-quantification method for the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) in urine was developed. The limit of detection was of 0.65 fmol (0.14 pg) injected or 65 pM. Heterocyclic aromatic amines are mutagenic and carcinogenic compounds formed at low levels in protein-rich food during cooking. Due to the low concentrations and the high complexity of the matrix in which they exist (food, blood, and urine), and the often small sample volumes (capillary blood; urine, blood and milk from small animals), very sensitive and selective analytical methods are required for their detection. Miniaturisation was accomplished by micro solid-phase extraction in capillaries with blue chitin as solid-phase, coupled on-line (in-capillary) to capillary electrophoresis with nanospray tandem mass spectrometric detection. This new technique provided a total analysis time of only 15 min, including extraction and separation, together with low sample and solvent consumption. Blue chitin showed high tolerance towards the urine matrix. Urine collected 12 h after consumption of fried chicken contained 1.8 nmol L −1 (0.40 pg μL −1) of PhIP.

Analytical methods in environmental effects-directed investigations of effluents

Effluent discharges are released into aquatic environments as complex mixtures for which there is commonly either no knowledge of the toxic components or a lack of understanding of how known toxicants interact with other effluent components. Effects-directed investigations consist of chemical extraction and iterative fractionation steps directed by a biological endpoint that is designed to permit the identification or characterization of the chemical classes or compounds in a complex mixture responsible for the observed biological activity. Our review of the literature on effects-directed analyses of effluents for non-mutagenic as well as mutagenic endpoints showed that common extraction and concentration methods have been used. Since the mid-1980s, the methods have evolved from the use of XAD resins to C18 solid-phase extraction (SPE). Blue cotton, blue rayon, and blue chitin have been used specifically for investigations of mutagenic activity where polycyclic compounds were involved or suspected. After isolation, subsequent fractionations have been accomplished using SPE or a high-pressure liquid chromatography (HPLC) system commonly fitted with a C18 reverse-phase column. Substances in active fractions are characterized by gas chromatography/mass spectrometry (GC-MS) and/or other spectrometric techniques for identification. LC-MS methods have been developed for difficult-to-analyze polar substances identified from effects-directed studies, but the potential for LC-MS to identify unknown polar compounds has yet to be fully realized. Salmonella-based assays (some miniaturized) have been coupled with fractionation methods for most studies aimed at identifying mutagenic fractions and chemical classes in mixtures. Effects-directed investigations of mutagens have focused mostly on drinking water and sewage, whereas extensive investigations of non-mutagenic effects have also included runoff, pesticides, and pulp mill effluents. The success of effects-directed investigations should be based on a realistic initial objective of each project. Identification of chemical classes associated with the measured biological endpoint is frequently achievable; however, confirmation of individual compounds is much more difficult and not always a necessary goal of effects-directed chemical analysis.

Blue Chitin Columns for the Extraction of Heterocyclic Amines from Urine Samples

During normal cooking of meat, a class of mutagenic/carcinogenic compounds called heterocyclic amines is formed. Heterocyclic amines are rapidly absorbed and metabolised in the human body, and for estimation of the intake of heterocyclic amines, it is useful to determinate their levels in the urine. Blue Chitin columns were used for the extraction and purification of heterocyclic amines from urine samples spiked with 14 different heterocyclic amines. The samples were analysed using LC-MS. The results show that Blue Chitin columns provide a straightforward and rapid means of extracting heterocyclic amines from urine samples, and that Blue Chitin column are also useful in the purification of urinary metabolites.

Blue chitin columns for the extraction of heterocyclic amines from cooked meat.

Mutagenic/carcinogenic heterocyclic amines (HAs) are formed at low levels (ng/g) during heat processing of protein-rich food such as meat and fish. The complex matrix requires effective extraction and purification methods. Blue Chitin columns were used for the extraction of HAs from fried chicken fillets and the samples were analysed with LC-MS. Several HAs were identified at levels ranging from 0.04 to 0.10 ng/g. The use of Blue Chitin columns provides a simple and fast method for the extraction of HAs from meat samples.

Mutagenic Characteristics and Contribution of Polycyclic Aromatic Hydrocarbons to Mutagenicity of Concentrates from Municipal River Water by Blue Chitin Column.

The mutagenic characteristics of concentrates from environmental water such as the effluent from various treatment plants and river waters using a modified Blue Chitin column method were determined by Ames assay with Salmonella typhimurium TA98, YG1021 and YG1024. This was done to estimate the quantitative contribution rate of mutagenicity estimated from analyzed polycyclic aromatic hydrocarbons (PAHs) content in the same concentrates to the mutagenicity of environmental water. Moreover, PAHs content and the mutagenicity of the river sediment-extract were measured to elucidate the fate of PAHs in the river. Many kinds of environmental waters and river sediment possessed principally indirect frame-shift type mutagenicity. Concentrates from many kinds of environmental waters possibly contained aminoarenes assayed with YG1021 and YG1024. In many kinds of environmental waters, the contribution rates of mutagenic magnitude estimated from seventeen analyzed PAH contents to the mutagenic magnitude of these water concentrates in TA98 with S9 mix were recognized to be from 0.10 to 1.15%. However, the contribution rate of mutagenic magnitude estimated from ten analyzed PAHs contents to the mutagenic magnitude of the Arata River sediment extract in TA98 with S9 mix was 64.2%. The high concentration of PAHs in the river sediment suggested that hydrophobic PAHs in the water might easily accumulate in the river sediment after adsorbing to the suspended solid.

ChemInform Abstract: Pigment Molecules Linked to Polymer Support: Blue Rayon, Blue Chitin, and Green Chitosan - Synthesis and Applications

The fact that hemin can inhibit the mutagenic activity of compounds bearing polycyclic structures is ascribable to the ability of the porphyrin structure to complex with the planar surface of the mutagens. The elucidation of this mechanism has led to the discovery of copper phthalocyanine trisulfonate (cpt) as an efficient ligand to trap polycyclic compounds on polymeric supports that bear cpt through covalent bond linkages. In blue cotton, the support for cpt is cotton, in blue rayon, it is amorphous rayon, and in blue chitin it is poly-N-acetylglucosamine. Using these blue materials, we have successfully isolated mutagens of polycyclic structures, e.g., heterocyclic amines, from environmental complex mixtures such as food, urine, feces, and river water. Preparation and properties of these adsorbents are described. Chlorophyllin linked to Sepharose and chitosan is also described. The use of these green materials is discussed.

ポリマー担体に固着した色素の反応 : ブルーレーヨン, ブルーキチン, グリーンキトサンなどの作製とその応用

The fact that hemin can inhibit the mutagenic activity of compounds bearing polycyclic structures is ascribable to the ability of the porphyrin structure to complex with the planar surface of the mutagens. The elucidation of this mechanism has led to the discovery of copper phthalocyanine trisulfonate (cpt) as an efficient ligand to trap polycyclic compounds on polymeric supports that bear cpt through covalent bond linkages. In blue cotton, the support for cpt is cotton, in blue rayon, it is amorphous rayon, and in blue chitin it is poly-N-acetylglucosamine. Using these blue materials, we have successfully isolated mutagens of polycyclic structures, e.g., heterocyclic amines, from environmental complex mixtures such as food, urine, feces, and river water. Preparation and properties of these adsorbents are described. Chlorophyllin linked to Sepharose and chitosan is also described. The use of these green materials is discussed.

A Fundamental Study on the Characteristics of Concentration Using a Blue Chitin Column for Polycyclic Aromatic Hydrocarbons in Water

In order to evaluate the contribution of polycyclic aromatic hydrocarbons (PAHs) to mutagenicity of concentrates from water, the adsorption, elution and recovery behavior of 26 PAHs bearing 3 to 6 rings were investigated by the Blue Chitin column method. The adsorption rates of 22 PAHs, having three or more fused rings, to Blue Chitin were over 90%. Elution rates of PAHs with 4 and 5 rings and nitroarenes (NO2-PAHs) adsorbed on Blue Chitin were 50-70% with 100ml of methanol-ammonia (CH3OH-NH4OH) (50 : 1). However, elution rates of PAHs with 3 and 6 rings and aminoarenes (NH2-PAHs) were 0-40% with 100ml of the same mixed eluate. However, additional elution with 20ml of dichloromethane (CH2Cl2) following the elution with 100ml of CH3OH-NH4OH (50 : 1) raised the elution ratio by 10-30%. Elution employing CH2Cl2 was thus more effective for elution of PAHs adsorbed to Blue Chitin. Using this preconcentration method on a modified Blue Chitin column, fluoranthene, pyrene, benz [a] anthracene, chrysene, benzo [b]-fluoranthene, benzo [k] fluoranthene, benzo [a] pyrene and dibenz [a, h] anthracene in effluent water and fluoranthene, pyrene and benzo [k] fluoranthene in river water were detected. In summary, this preconcentration method using a modified Blue Chitin column was useful for concentration of PAHs and NO2-PAHs with 4 and 5 rings from environmental water.

〈第５回生物利用新技術研究シンポジウム〉より ブルーキチン・カラム／芳香族アミン高感受性株を用いた水域環境の変異原性モニタリング

〈第５回生物利用新技術研究シンポジウム〉より ブルーキチン・カラム／芳香族アミン高感受性株を用いた水域環境の変異原性モニタリング