[Screening of risky substances in sea cucumbers based on gas chromatography-orbitrap high-resolution mass spectrometry].

A new method for sample pretreatment using improved QuEChERs was established, and 289 organic pollutants with health risks could be identified and quantified through gas chromatography-orbitrap high-resolution mass spectrometry (GC-Orbitrap HRMS). A high-resolution database of 289 environmental pollutants belonging to ten categories, including organochlorine pesticides (OCPs), polycyclic aromatic hydrocarbons (PAHs), phthalates (PAEs), polychlorinated biphenyls (PCBs), and other agricultural chemicals (ACs), was established for the non-targeted screening and quantitative analysis. A simple method for biological sample preparation using improved QuEChERs was proposed by combining a conventional QuEChERs method and a column purification method. After purification using a Florisil column, the lipid content was reduced by 99.9%, which significantly reduced the interference of the matrix effect observed during the analysis. Furthermore, simultaneous high-accuracy qualitative screening and quantitative analysis of the target compounds were performed through high-resolution mass spectrometry (60000 resolution) conducted in the full scan mode. The limits of quantification were 0.56-57.8 pg/g, presenting a large linear range (~106), and the recovery range was 40%-120%. Due to the high-resolution and sensitivity of Q Exactive GC-Orbitrap HRMS, the limits of quantification of the target compounds were significantly lower than those achieved through methods based on conventional chromatography and mass spectrometry. Moreover, ultratrace organic contaminants that cannot be detected by conventional methods can be accurately quantified by the proposed method. Sea cucumber samples collected at the breeding site were analyzed using the proposed high-coverage multi-objective analytical method, and more than 100 types of organic pollutants were detected; the mean contents of PAHs, ACs, PAEs, and OCPs were 157.8, 153.2, 64.4, and 46.4 ng/g dw, respectively, which were higher than those of other pollutants. Some new contaminants, such as 9-chlorofluorene, 5-chloroacenaphthene, and 3-methylcholanthrene, were detected at very low contents for the first time in the sea cucumber samples. The proposed method is simple and efficient, allows the detection of pollutants at very low contents, and provides accurate and reliable results. Thus, this high-coverage multi-objective analytical method can be widely used for broad-spectrum screening and accurate quantification of contaminants in various aquatic products, providing technical support for food safety control.

Microfluidic geometric metering-based multi-reagent mixture generator for robust live cell screening array.

Microfluidic live cell arrays with integrated concentration gradient or mixture generators have been utilized in screening cellular responses to various biomolecular cues. Microfluidic network-based gradient generators that can create concentration gradients by repeatedly splitting and mixing different solutions using networks of serpentine channels are commonly used. However, in this method the generation of concentration gradients relies on the continuous flow of sample solutions at optimized flow rates, which poses challenges in maintaining the pressure and flow stability throughout the entire assay period. Here we present a microfluidic live cell screening array with an on-demand multi-reagent mixture generator where the mixing ratios, thus generated concentrations, are hard-wired into the chip itself through a geometric metering method. This platform showed significantly improved robustness and repeatability in generating concentration gradients of fluorescent dyes (average coefficient of variance C.V. = 9 %) compared to the conventional network-based gradient generators (average C.V. = 21 %). In studying the concentration dependent effects of the environmental toxicant 3-methylcholanthrene (3MC) on the activation of cytochrome P450 1A1 (Cyp 1A1) enzyme in H4IIE rat hepatoma cells, statistical variation of the Cyp 1A1 response was significantly lower (C.V. = 5 %) when using the developed mixture generator compared to that using the conventional gradient generator (C.V. = 12 %). Reduction in reagent consumption by 12-times was also achieved. This robust, accurate, and scalable multi-reagent mixture generator integrated with a cell culture array as a live cell assay platform can be readily implemented into various screening applications where repeatability, robustness, and low reagent consumptions over long periods of assay time are of importance.

Fast-track DRESSA: a bioassay for fast, sensitive, and selective detection of halogenated and polycyclic aromatic hydrocarbons.

Dioxin and related chemicals cause a variety of toxic and biological effects via the aryl hydrocarbon receptor (AhR). We recently reported a mammalian cell-based bioassay system (dioxin-responsive-element-based sensing via secreted alkaline phosphatase; DRESSA) that can detect dioxin and dioxin-like chemicals with high sensitivity. In this report, we describe an advanced method (designated "fast-track DRESSA") that achieves fast, selective, and sensitive detection of dioxin and other toxic compounds. By optimization of assay conditions on cell number and serum concentration, the fast-track DRESSA enabled detection of 0.5 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin within 6 h. It also enabled detection of 10 pM 3-methylcholanthrene, 100 pM benzo[a]pyrene, and 100 pM beta-naphthoflavone within 6-16 h. By combination with the AhR antagonist alpha-naphthoflavone, nonspecific, false-positive responses could be eliminated. Because of its time-saving property and easiness, sensitiveness, and specificity, the fast-track DRESSA would be advantageous for high-throughput screening of dioxin and dioxin-like compounds in environmental samples.

Evanescent fluorobiosensor for the detection of polyaromatic hydrocarbon based on DNA intercalation.

A flow-injection analysis (FIA) system coupled with an evanescent wave (EW) biosensor employing total internal reflection of fluorescence radiation (TIRF) for the detection of polyaromatic hydrocarbon that intercalates into DNA is reported. A highly fluorescent intercalator, "ethidium bromide," has been used as the reference compound for detection. The EW biosensor was developed according to the procedure described earlier (1,2). Data on the analysis of Naphthalene, 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene, 1,2-benzanthracene, and some standard reference materials supplied by the National Institute of Standards and Technology are reported. The relative ability of the polyaromatic hydrocarbon to displace ethidium bromide, based on the relative binding ratio, is found to be on the order of 7,12-dimethylbenz[a]anthracene > 3-methylcholanthrene > 1,2-benzanthracene > napthalene.

[Recent progress in studies of the drug-metabolizing enzyme cytochrome P-450].

More than a dozen nearly full-length cDNA clones for cytochromes P-450 were isolated from various sources. From their nucleotide sequences, the primary structures of the corresponding proteins were deduced. All the P-450s determined showed statistically significant sequence homology among them. On the basis of this homology, the molecular evolution of the P-450 super gene family was discussed, and the functional domains of the proteins including the heme-binding site were estimated. Chromosomal DNAs for methylcholanthrene- and phenobarbital-inducible P-450s, the two inducible forms of P-450 which diverged approximately 400 million years ago, were cloned, and their gene structures were analyzed. It was interesting that all the exon-intron boundaries between the two subgene families were found not to be conserved. The mechanism of inductive expression of the methylcholanthrene-inducible P-450 c that catalyzes detrimental reactions of chemical carcinogens and mutagens such as benzo[a]pyrene were investigated by the method of DNA transfection. Several cis-acting DNA elements responsible for the regulation were located in the 5' flanking region of the P-450 c gene.

Direct enantiomeric resolution of cyclic alcohol derivatives of polycyclic aromatic hydrocarbons by chiral stationary phase high-performance liquid chromatography.

The enantiomers of some cyclic alcohol derivatives of phenanthrene, benz[a]anthracene, benzo[a]pyrene, cholanthrene, and 3-methylcholanthrene were resolved by high-performance liquid chromatography using a commercially available preparative column packed with an (R)-N-(3,5-dinitrobenzoyl)phenylglycine ionically bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption, mass and circular dichroism spectral analyses. This method has been applied to the determination of optical purity of 1-hydroxycholanthrene and 1-hydroxy-3-methylcholanthrene formed in the metabolism of cholanthrene and 3-methylcholanthrene, respectively, by rat liver microsomes.

A method for measurement of nanogram quantities of 3-methylcholanthrene in stool samples.

The carcinogen 3-methylcholanthrene can be produced from deoxycholic acid and is postulated by some investigators to play a role in the pathogenesis of colon carcinoma. The small quantities of this compound which could be carcinogenic have been difficult to measure in feces because of many potentially interfering compounds. Using 3-[6-14C]methylcholanthrene as an internal standard, petroleum ether extraction, C-18 SepPak separation, preparative high performance liquid chromatography, and gas-liquid chromatography-mass spectrometry with selected ion monitoring, we developed an assay capable of detecting less than 35 ng of 3-methylcholanthrene per gram of stool. Application of this technique to stools of five patients with colon carcinoma and two normal controls revealed no detectable 3-methylcholanthrene in any stool sample. This negative result was confirmed by incubating radiolabeled cholic acid in fecal homogenates. Although greater than 90% of this radiolabeled bile acid was converted to deoxycholic acid, none of the radioactivity was found in the thin-layer chromatography fraction corresponding to 3-methylcholanthrene. These observations provide evidence against a role for 3-methylcholanthrene in pathogenesis of human colon carcinoma. Similar assays could be used for analysis of other carcinogens in stool samples.

Effect of diet on fecal excretion and gastrointestinal tract distribution of unmetabolized benzo(a)pyrene and 3- methylcholanthrene when these compounds are administered orally to hamsters.

3-Methylcholanthrene (3MC) administered p.o. has induced tumors of the hamster gastrointestinal tract (GIT), including the large intestine. This process may depend on the concentration of unchanged hydrocarbon in the GIT contents. Benzo(a)pyrene (BP) ingestion could be involved in human GIT carcinogenesis. Accordingly, male Syrian golden hamsters were fed diets containing BP or 3MC for 10 days. Feces collected during the last two to three days of feeding were analyzed for the unchanged hydrocarbons by KOH:methanol digestion, Florisil column and paper chromatography, and ultraviolet spectrophotometry. With a semisynthetic diet containing 5% Alphacel, 6% corn oil, and 100 microgram BP per g. fecal BP excretion was 0.45% of the dose. Variation of the corn oil content had little effect. Fecal BP excretion was increased 13 times (to 6% of the dose) when 5% wheat brain was used in place of Alphacel and 4.5 times when a commercial diet was used. This suggests that bran adsorbed or sequestered the BP. Water content of the large-intestine contents was increased when the brain diet was fed. Both these factors could affect mucosal exposure to BP. For 3MC, fecal excretion of unchanged hydrocarbon was 14 times greater than for BP under similar conditions. The GIT contents of hamsters fed BP or 3MC showed hydrocarbon concentrations in the order: stomach greater than lower large intestine greater than other sections.

The initiation of tumours on mouse skin by dihydrodiols derived from 7,12-dimethylbenz(a)anthracene and 3-methylcholanthrene.

The cis-2a,3-diol and the trans-4,5-, trans-7,8-, trans-9,10- and trans-11,12-dihydrodiols of 3-methylcholanthrene and the trans-3,4, trans-5,6-, trans-8,9. and trans-10,11- dihydrodiols of 7,12-dimethylbenz[a]anthrancene have been tested, in comparison with the parent hydrocarbons, for their abilities to initiate skin tumours in female CDI mice. Groups of mice received a single topical application (25 micrograms) of a diol or of a hydrocarbon, and 1 week later repeated topical applications (1 microgram) of 12-0-tetradecanoylphorbol-13-acetate were commenced. The results show that the diol of 3-methylcholanthrene and the 3,4-diol of 7,12-dimethylbenz[a]anthrancene were active as initiating agents but that they were no more active than their parent hydrocarbon. The K-region 5,6-diol of 7,12-dimethylbenz[a]anthrancene, which cannot be converted directly into a vicinal diol-epoxide, was also active as a tumour-initiating agent when applied to mouse skin.

[On the presence of 20-methylcholanthrene in the atmospheric air (author's transl)]. / Uber das Vorhandensein des 20-Methylcholanthrens in der atmosphärischen Luft.

The strongly carcinogenic polycyclic aromatic hydrocarbon 20-Methylcholanthrene was detected in the urban atmospheric air in Budapest (the capital of Hungary). The main source of this pollution seems to be the motor vehicle traffic, since samples from a heavy traffic junction contained up to six times higher 20-Methylcholanthrene concentrations than samples from a low traffic area. Ultraviolet absorption spectrophotometry yielded similar results. The presence of 20-Methylcholanthrene is apparently connected with the presence of 3,4-Benzpyrene and 1,2-Benzpyrene, respectively.

Morphological transformation of early passage golden Syrian hamster embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse carcinogens.

Cryopreserved primary cultures of golden Syrian hamster embryo cells were used as the source of target and feeder cells for establishing an in vitro carcinogenesis bioassay. The primary culture giving the best overall response in a pretest before freezing gave positive results in 20 consecutive experiments when retested with 3-methylcholanthrene after cryopreservation, indicating that pretested cryopreserved cultures can serve as a source of susceptible target cells in an in vitro carcinogenesis bioassay. Similarly prepared and cryopreserved cultures served satisfactorily as feeder cells. Susceptible positive cultures were used to test a large number of carcinogenic and non-carcinogenic chemicals in this system. The results showed a very high positive correlation (90.8%) between morphological transformation and the reported carcinogenic activity of the chemicals. Transformation was not observed when cells were tested with a few carcinogens that may not be metabolized to their active forms by early passage hamster embryo cells. N-2-acetylaminofluorene transformed cells only when tested in the presence of hamster liver microsomes. No false positive results were obtained when non-carcinogens were bioassayed, nor was spontaneous transformation observed in control cultures treated with medium alone, 0.2% dimethylsulfoxide or other solvents. Cultures derived from morphologically transformed colonies arising after treatment of cells with several known carcinogens were tumorigenic in vivo, confirming the correlation of morphological transformation with tumorigenicity and the validity of altered morphology as an in vitro criterion for carcinogenicity in vivo.