Integrative Inducer Intervention and Transcriptomic Analyses Reveal the Metabolism of Paralytic Shellfish Toxins in Azumapecten farreri.

Paralytic shellfish toxins (PSTs) are widely distributed neurotoxins, and the PST metabolic detoxification mechanism in bivalves has received increasing attention. To reveal the effect of phase I (cytochrome P450)-II (GST)-III (ABC transport) metabolic systems on the PST metabolism in Azumapecten farreri, this study amplified stress on the target systems using rifampicin, dl-α-tocopherol, and colchicine; measured PST levels; and conducted transcriptomic analyses. The highest toxin content reached 1623.48 µg STX eq/kg in the hepatopancreas and only 8.8% of that in the gills. Inducer intervention significantly decreased hepatopancreatic PST accumulation. The proportional reductions in the rifampicin-, dl-α-tocopherol-, and colchicine-induced groups were 55.3%, 50.4%, and 36.1%, respectively. Transcriptome analysis showed that 11 modules were significantly correlated with PST metabolism (six positive/five negative), with phase I CYP450 and phase II glutathione metabolism significantly enriched in negatively correlated pathways. Twenty-three phase I-II-III core genes were further validated using qRT-PCR and correlated with PST metabolism, revealing that CYP46A1, CYP4F6, GSTM1, and ABCF2 were significantly correlated, while CYP4F11 and ABCB1 were indirectly correlated. In conclusion, phase I-II-III detoxification enzyme systems jointly participate in the metabolic detoxification of PSTs in A. farreri. This study provides key data support to profoundly elucidate the PST metabolic detoxification mechanism in bivalves.

Phenylhydrazone Alkaloids from the Deep-Sea Cold Seep Derived Fungus Talaromyces amestolkiae HDN21-0307.

Alkaloids with a phenylhydrazone architecture are rarely found in nature. Four unusual phenylhydrazone alkaloids named talarohydrazones A-D (1-4) were isolated from the deep-sea cold seep derived fungus Talaromyces amestolkiae HDN21-0307 using the one strain-many compounds (OSMAC) approach and MS/MS-based molecular networking (MN) combined with network annotation propagation (NAP) and the unsupervised substructure annotation method MS2LDA. Their structures were elucidated by spectroscopic data analysis, single-crystal X-ray diffraction, and quantum chemical calculations. Talarohydrazone A (1) possessed an unusual skeleton combining 2,4-pyridinedione and phenylhydrazone. Talarohydrazone B (2) represents the first natural phenylhydrazone-bearing azadophilone. Bioactivity evaluation revealed that compound 1 exhibited cytotoxic activity against NCI-H446 cells with an IC50 value of 4.1 µM. In addition, compound 1 displayed weak antibacterial activity toward Staphylococcus aureus with an MIC value of 32 µg/mL.

Metabolites and metabolic pathway analysis of sulfadimidine in carp (Cyprinus carpio) based on UHPLC-Q-orbitrap HRMS.

Sulfadimidine (SM2) is an N-substituted derivative of p-aminobenzenesulfonyl structure. This study aimed to analyze the metabolism of SM2 in carp (Cyprinus carpio). The carps were fed with SM2 at a dose of 200 mg/(kg · bw) and then killed. The blood, muscle, liver, kidney, gill, other guts, and carp aquaculture water samples were collected. The UHPLC-Q-Exactive Plus Orbitrap-MS was adopted for determining the metabolites of SM2 in the aforementioned samples. Twelve metabolites, which were divided into metabolites in vivo and metabolites in vitro, were identified using Compound Discoverer software. The metabolic pathways in vivo of SM2 in carp included acetylation, hydroxylation, glucoside conjugation, glycine conjugation, carboxylation, glucuronide conjugation, reduction, and methylation. The metabolic pathways in vitro included oxidation and acetylation. This study clarified the metabolites and metabolic pathways of SM2 in carp and provided a reference for further pharmacodynamic evaluation and use in aquaculture.

Contamination Status and Risk Assessment of Paralytic Shellfish Toxins in Shellfish along the Coastal Areas of China.

Paralytic shellfish toxins (PSTs) are widely distributed in shellfish along the coast of China, causing a serious threat to consumer health; however, there is still a lack of large-scale systematic investigations and risk assessments. Herein, 641 shellfish samples were collected from March to November 2020, and the PSTs' toxicity was detected via liquid chromatography-tandem mass spectrometry. Furthermore, the contamination status and potential dietary risks of PSTs were discussed. PSTs were detected in 241 shellfish samples with a detection rate of 37.60%. The average PST toxicities in mussels and ark shells were considerably higher than those in other shellfish. The PSTs mainly included N-sulfonylcarbamoyl toxins (class C) and carbamoyl toxins (class GTX), and the highest PST toxicity was 546.09 µg STX eq. kg-1. The PST toxicity in spring was significantly higher than those in summer and autumn (p < 0.05). Hebei Province had the highest average PST toxicity in spring. An acute exposure assessment showed that consumers in Hebei Province had a higher dietary risk, with mussels posing a significantly higher dietary risk to consumers. This research provides reference for the green and sustainable development of the shellfish industry and the establishment of a shellfish toxin prevention and control system.

Dimeric Tetracenomycin Derivatives from a Taklamakan Desert-Derived Streptomyces sp. HDN154193.

Bitetracenomycin A (1) and its diastereomers [(±)-bitetracenomycin B, (±)-2] were discovered from the cultures of Streptomyces sp. HDN154193. Compounds 1 and (±)-2 were the first tetracenomycin dimers obtained from a natural source with sp3 methine protons at the bridge positions (C-12/12'), which also exhibited broad-spectrum antibacterial activity. The racemate (±)-2 was semisynthesized and separated into enantiomers (+)-2 and (-)-2, and the absolute configurations were determined by specific rotation and ECD data. These metabolites exhibited potent antibacterial activity especially against drug-resistant strains (MRSA and MRCNS) with MIC values ranging from 1.0 to 1.9 µg/mL.

Simultaneous determination of triazine herbicides and their metabolites in shellfish by HPLC-MS/MS combined with Q/E-Orbitrap HRMS.

Triazine herbicides are used extensively in agriculture and aquaculture worldwide because of their broad effectiveness in weed control. However, after they are discharged into the sea, they seriously contaminate aquatic ecosystems and threaten aquatic organisms, especially shellfish. Currently, there are no established methods for the detection and confirmation of triazine herbicides and their metabolites in biological matrixes. Hence, the food safety of aquatic products cannot be accurately evaluated, which creates a technical barrier against international aquatic product trade. In this study, for the first time, a method was developed for the analysis and confirmation of seven triazine herbicides and 13 metabolites in shellfish, based on alkaline acetonitrile extraction and neutral Al2O3 cartridge purification coupled with internal standard calibration. Specifically, quantitative and qualitative analysis was conducted using high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS/MS), and accurate identification was carried out by quadrupole orbitrap high-resolution mass spectrometry (Q/E Orbitrap HRMS). The results showed that target analytes demonstrated good linearity within the corresponding range (R2 > 0.995). The limit of detection and limit of quantitation of the proposed method were 0.1 and 0.3 µg/kg, respectively. The average recoveries of analytes were between 70.0% and 120% when spiked at three levels with blank oyster (Crassostrea gigas) as the matrix, and the relative standard deviations (RSDs) were all less than 12% (n=6). The proposed method was successfully applied for the detection of triazine herbicide residues in oyster samples during actual breeding, and the presence of DIP, HP, DEHA, and other metabolites in positive samples was confirmed by Q/E Orbitrap HRMS. This method exhibits high accuracy, high sensitivity, and good reproducibility. It has promising application prospects in the field of hazard analysis and the positive identification of aquatic products.

Antibacterial p-Terphenyl with a Rare 2,2'-Bithiazole Substructure and Related Compounds Isolated from the Marine-Derived Actinomycete Nocardiopsis sp. HDN154086.

Assisted by MS/MS-based molecular networking and X-ray diffraction analysis, five new p-terphenyl derivatives, namely, nocarterphenyls D-H (1-5), were obtained and characterized from the cultures of the marine sediment-derived actinomycete Nocardiopsis sp. HDN154086. The skeleton of nocarterphenyl D (1) was defined to possess a rare 2,2'-bithiazole scaffold, naturally occurring for the first time, and nocarterphenyls E-H (2-5) are p-terphenylquinones with unusual thioether linked fatty acid methyl ester substitutions. Compound 1 showed promising activity against multiple bacteria with MIC values ranging from 1.5 to 6.2 µM, and 2 exhibited notable antibacterial activity against MRSA which surpassed the positive control ciprofloxacin.

Effects of single and combined exposure to BDE-47 and PFOA on distribution, bioaccumulation, and toxicity in blue mussel (Mytilus galloprovincialis).

The contamination of 2,2',4,4'-Tetrabrmodiphenyl ether (BDE-47) and perfluorooctanoic acid (PFOA) has drawn a worldwide attention over the risks in ecological and food safety. In this work, blue mussel (Mytilus galloprpvincialis) was employed to investigate the combined effects of BDE-47 (10 ng mL-1) and PFOA (100 ng mL-1) on tissue distribution, accumulation, elimination, and toxicity. Results suggested that BDE-47 and PFOA accumulated mostly in digestive gland, followed by gills and gonad, and M. galloprovincialis displayed higher accumulation capacity to BDE-47 than PFOA. Co-exposure treatment reduced the accumulation of BDE-47, and enhanced the accumulation of PFOA. Furthermore, biochemical and histopathological tests revealed that the aggravated toxicity in co-exposure groups was mainly attributed to the oxidative stress and damage of tissue structure. This work could be helpful to get a better understanding of the combined behaviors and cumulative risks of BDE-47 and PFOA in marine ecosystem.

Nontarget Screening and Toxicity Evaluation of Diol Esters of Okadaic Acid and Dinophysistoxins Reveal Intraspecies Difference of Prorocentrum lima.

High-resolution mass spectrometry (HRMS) analysis with the assistance of molecular networking was used to investigate intracellular toxin profiles of five Prorocentrum lima (P. lima) strains sampled from the north Yellow Sea and South China Sea. Mice were used as a model species for testing the acute toxicity of intracellular okadaic acid (OA) and dinophysistoxins (DTXs) in free and esterified states. Results showed that OA and DTX1 esterified derivatives were detected in all P. lima samples, accounting for 55%-96% of total toxins in five strains. A total of 24 esters and 1 stereoisomer of DTX1 (35S DTX1) were identified based on molecular networking and MS data analysis, 15 esters of which have been reported first. All P. lima strains displayed specific toxin profiles, and preliminary analysis suggested that toxin profiles of the five P. lima strains might be region-related. Moreover, acute toxicity in mice suggested higher toxicity of esters compared with free toxins, which highlights the importance and urgency of attention to esterified toxins in P. lima.

Secondary Metabolites Produced by Combined Culture of Penicillium crustosum and a Xylaria sp.

Four new alkyl aromatics, penixylarins A-D (1-4), along with the known biogenetically related 1,3-dihydroxy-5-(12-hydroxyheptadecyl)benzene (5) and 1,3-dihydroxy-5-(12-sulfoxyheptadecyl)benzene (6), were isolated from a mixed culture of the Antarctic deep-sea-derived fungus Penicillium crustosum PRB-2 and the mangrove-derived fungus Xylaria sp. HDN13-249. UPLC-MS data and an analysis of structural features showed that compounds 1 and 2 were produced by collaboration of the two fungi, while compounds 3-6 could be produced by Xylaria sp. HDN13-249 alone, but in noticeably increased quantities by cocultivation. Compounds 2, 3, 5, and 6 showed antibacterial activity against a panel of strains, and compound 3 possessed potential antituberculosis effects (MIC = 6.25 µM against Mycobacterium phlei).

Selective enrichment and quantification of okadaic acid in shellfish using an immunomagnetic-bead-based liquid chromatography with tandem mass spectrometry assay.

Okadaic acid is a marine biotoxin that primarily occurs in shellfish and can cause diarrheic shellfish poisoning in humans. When analyzing biological samples using liquid chromatography with tandem mass spectrometry, the presence of complex matrices is a major issue. Thus, it is crucial to selectively and simply extract the target analyte from samples and minimize matrix effects simultaneously. To meet this need, an immunomagnetic-bead-based liquid chromatography with tandem mass spectrometry method was developed to detect okadaic acid in shellfish. Magnetic beads bound to monoclonal antibody against okadaic acid were used as affinity probes to specifically enrich okadaic acid in samples, which effectively eliminated matrix effects. A magnetic separator was used to aggregate and separate magnetic particles from sample matrices, and methanol was used to elute okadaic acid from the magnetic beads. Standard solution prepared with methanol was employed directly for quantitative analysis. Several experimental conditions were optimized to improve performance. The method is of interest as a rapid (10 min) sample clean-up and selective enrichment tool, and it showed good linearity and sensitivity, with reported limits of detection and quantitation of 3 and 10 µg/kg, respectively. Fifty-three shellfish samples from an aquatic products market were tested using this method, and four samples positive for okadaic acid were found.

Anthraquinone Derivatives from a Marine-Derived Fungus Sporendonema casei HDN16-802.

Five new anthraquinone derivatives, auxarthrols D-H (1-5), along with two known analogues (6-7), were obtained from the culture of the marine-derived fungus Sporendonema casei. Their structures, including absolute configurations, were established on the basis of NMR, HRESIMS, and circular dichroism (CD) spectroscopic techniques. Among them, compound 4 represents the second isolated anthraquinone derivative with a chlorine atom, which, with compound 6, are the first reported anthraquinone derivatives with anticoagulant activity. Compounds 1 and 3 showed cytotoxic activities with IC50 values from 4.5 µM to 22.9 µM, while compounds 1, 3-4, and 6-7 showed promising antibacterial activities with MIC values from 12.5 µM to 200 µM. In addition, compound 7 was discovered to display potential antitubercular activity for the first time.

New Glutamine-Containing Azaphilone Alkaloids from Deep-Sea-Derived Fungus Chaetomium globosum HDN151398.

Three new azaphilone alkaloids containing glutamine residues, namely N-glutarylchaetoviridins A-C (1-3), together with two related compounds (4 and 5) were isolated from the extract of Chaetomium globosum HDN151398, a fungus isolated from a deep-sea sediment sample collected in South China Sea. Their structures were elucidated on the basis of extensive 1D and 2D NMR as well as HRESIMS spectroscopic data and chemical analysis. N-glutarylchaetoviridins A-C (1-3) represent the first class of chaetoviridins characterized by embedded glutamate residues. Amino acids incubation experiments produced five azaphilone laden different amino acids residues (6-10) which indicated that this method can enhanced the structural diversity of this strain by culturing with amino acids. Cytotoxicity of the isolated compounds were evaluated against a panel of human cancer cell lines.

Distribution of perfluorinated alkyl substances in marine shellfish along the Chinese Bohai Sea coast.

Perfluorinated alkyl substances (PFASs) are emerging persistent organic pollutants, which pose a threat to human health primarily by dietary exposure, especially through seafood. Bohai Sea (a semi-closed sea located north of China) is an important shellfish aquaculture area that is possibly highly-polluted with PFASs. In this study, we first evaluated contamination by PFASs in a total of 230 samples of marine shellfish from the Bohai Sea. Samples included five important shellfish species, collected from important aquaculture spots distributed around the Bohai Sea. Samples were analyzed by an ultra-fast liquid chromatography-tandem mass spectrometry method, which could simultaneously detect 23 PFASs in shellfish. Our research verified that PFASs have become a threat to the safety of shellfish products in this area. Furthermore, contamination by PFASs in shellfish changed depending on the components of PFASs, the species of shellfish, and the sampling sites. Many of the 23 target compounds contributed to the high detection ratio (>50%) as follows: perfluorooctanoic acid (PFOA) > perfluorononanoic acid > perfluorodecanesulfonic acid > perfluorooctanesulfonic acid (PFOS). Compared with other dominant components, PFOA not only had the highest detection percentage in shellfish samples (98.3%), but its detection level contributed to 87.2% of total PFASs concentrations, indicating that PFOA is the major threat to the safety of shellfish products. The highest level of PFAS was found in clams (62.5 ng g-1 wet weight of PFOA). The concentration of total PFAS in different shellfish species showed the following trend: clams > mussels > scallops > whelks > oysters. The maximum concentration of total PFAS or PFOA was found in Shouguang. The total concentration of PFOS and its precursor were highest in Cangzhou, possibly due to local industrial activities. The results presented in this paper provide new data on the contamination of marine shellfish along the Bohai Sea coasts in China, and constitute a reference for future monitoring of contamination by emerging contaminants in Bohai coast.

Austalides S-U, New Meroterpenoids from the Sponge-Derived Fungus Aspergillus aureolatus HDN14-107.

Three new meroterpenoids, named austalides S-U (1-3), were isolated from the culture of a sponge-derived fungus Aspergillus aureolatus HDN14-107, together with eleven known austalides derivates (4-14). Their structures, including absolute configurations, were assigned on the basis of NMR, MS data, and TDDFT ECD calculations. Compound 1 is the first case of austalides with the terpene ring fused to the chroman ring in trans configuration. Compounds 3 and 5 exhibited activities against influenza virus A (H1N1), with IC50 values of 90 and 99 µM, respectively.

Penicitols A-C and penixanacid A from the mangrove-derived Penicillium chrysogenum HDN11-24.

Three new citrinin analogues, penicitols A-C (1-3), and one new xanthone derivative, penixanacid A (4), together with four known biogenetically related compounds (5-8), were discovered from the extract of a mangrove-derived fungus, Penicillium chrysogenum HND11-24. The structures of penicitols A-C and penixanacid A were established through analysis of extensive spectroscopic data. Their cytotoxic activity against HeLa, BEL-7402, HEK-293, HCT-116, and A549 cell lines was evaluated.

Versixanthones A-F, Cytotoxic Xanthone-Chromanone Dimers from the Marine-Derived Fungus Aspergillus versicolor HDN1009.

Six unusual xanthone-chromanone dimers, versixanthones A-F (1-6), featuring different formal linkages of tetrahydroxanthone and 2,2-disubstituted chroman-4-one monomers, were isolated from a culture of the mangrove-derived fungus Aspergillus versicolor HDN1009. The absolute configurations of 1-6, representing the central and axial chirality elements or preferred helicities, were established by a combination of X-ray diffraction analysis, chemical conversions, and TDDFT-ECD calculations. The interconversion of different biaryl linkages between 1 and 4 and between 2 and 3 in DMSO by a retro-oxa-Michael mechanism provided insight into the formation of the xanthone-chromanone dimers and supported the assignments of their absolute configurations. Compounds 1-6 exhibited cytotoxicities against the seven tested cancer cell lines, with the best IC50 value of 0.7 µM. Compound 5 showed further inhibitory activity against topoisomerase I.

Rare Chromones from a Fungal Mutant of the Marine-Derived Penicillium purpurogenum G59.

Three new and rare chromones, named epiremisporine B (2), epiremisporine B1 (3) and isoconiochaetone C (4), along with three known remisporine B (1), coniochaetone A (5) and methyl 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (6) were isolated from a mutant from the diethyl sulfate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. The structures of 2-4 including the absolute configurations were determined by spectroscopic methods, especially by NMR analysis and electronic circular dichroism (ECD) experiments in conjunction with calculations. The absolute configuration of the known remisporine B (1) was determined for the first time. Compounds 2 and 3 have a rare feature that has only been reported in one example so far. The compounds 1-6 were evaluated for their cytotoxicity against several human cancer cell lines. The present work explored the great potential of our previous DES mutagenesis strategy for activating silent fungal pathways, which has accelerated the discovery of new bioactive compounds.

Prenylated indole diketopiperazines from the marine-derived fungus Aspergillus versicolor.

Seven new prenylated indole diketopiperazines, versicamides A-G (1-7) and a novel chemical derivative from 7, versicamide H (8), along with three known analogic diketopiperazines (9-11) were obtained from the marine-derived fungus Aspergillus versicolor HDN08-60. Their structures were determined by spectroscopic techniques, including 2D NMR, ECD calculations, and single-crystal X-ray diffraction analysis, together with the assistance of further chemical conversions. The cytotoxicities of 1-8 were tested against the HeLa, HCT-116, HL-60, and K562 cell lines, but only 8 exhibited moderate activity against HL-60 cells, with an IC50 value of 8.7 µM. Further investigation with target screening showed that 8 exhibited selective PTK inhibitory activities.

Sorbicatechols A and B, antiviral sorbicillinoids from the marine-derived fungus Penicillium chrysogenum PJX-17.

Two novel sorbicillinoids combining a bicyclo[2.2.2]octane with a 2-methoxyphenol moiety, named sorbicatechols A (1) and B (2), were isolated from the culture of the marine sediment-derived fungus Penicillium chrysogenum PJX-17, together with the known protocatechuic acid methyl ester and caffeic acid methyl ester (3). Their structures, including absolute configurations, were assigned by analysis of NMR, MS data, and TDDFT ECD calculations. Compounds 1 and 2 exhibited activities against influenza virus A (H1N1), with IC50 values of 85 and 113 µ M, respectively.