Assessment of estrogenic potential from exudates of microcystin-producing and non-microcystin-producing Microcystis by metabolomics, machine learning and E-screen assay.

Cyanobacterial blooms, often dominated by Microcystis aeruginosa, are capable of producing estrogenic effects. It is important to identify specific estrogenic compounds produced by cyanobacteria, though this can prove challenging owing to the complexity of exudate mixtures. In this study, we used untargeted metabolomics to compare components of exudates from microcystin-producing and non-microcystin-producing M. aeruginosa strains that differed with respect to their ability to produce microcystins, and across two growth phases. We identified 416 chemicals and found that the two strains produced similar components, mainly organoheterocyclic compounds (20.2%), organic acids and derivatives (17.3%), phenylpropanoids and polyketides (12.7%), benzenoids (12.0%), lipids and lipid-like molecules (11.5%), and organic oxygen compounds (10.1%). We then predicted estrogenic compounds from this group using random forest machine learning. Six compounds (daidzin, biochanin A, phenylethylamine, rhein, o-Cresol, and arbutin) belonging to phenylpropanoids and polyketides (3), benzenoids (2), and organic oxygen compound (1) were tested and exhibited estrogenic potency based upon the E-screen assay. This study confirmed that both Microcystis strains produce exudates that contain compounds with estrogenic properties, a growing concern in cyanobacteria management.

Impaired intestinal immunity and microbial diversity in common carp exposed to cadmium.

Waterborne cadmium (Cd) accumulates in the fish intestine and causes irreversible toxicity by disrupting intestinal immunity and microbial diversity. To explore the toxicity of environmentally available high Cd concentration on intestinal immunity and microbial diversity of fish, we selected the widely used bioindicator model species, Common carp (Cyprinus carpio). Literature review and Cd pollution data supported sequential doses of 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/L Cd for 30 days. Based on intestinal tissue Cd accumulation, previous studies, and environmentally available Cd data, 0.4 and 1.6 mg/L Cd were selected for further studies. Intestinal Cd bioaccumulation increased significantly to ~100 times in fish exposed to 1.6 mg/L Cd. We observed villous atrophy, increased goblet cells with mucus production, muscularis erosion, and thickened lamina propria due to intense inflammatory cell infiltration in the intestine at this Cd concentration. Cd-induced immunosuppression occurred with increased lysozyme, alkaline phosphate (AKP), and acid phosphate (ACP). High levels of catalase (CAT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), and hydrogen peroxide (H2O2) suggested induced oxidative stress and poor metabolism by α-amylase and lipase suppression for Cd toxicity. Proteobacteria (41.2 %), Firmicutes (21.8 %), and Bacteroidetes (17.5 %) were the dominant bacterial phyla in the common carp intestine. Additionally, potential pathogenic Cyanobacteria increased in Cd-treated fish. The decrease of beneficiary bacteria like Aeromonas, and Cetobacterium indicated Cd toxicity. Overall, these findings indicate harmful consequences of high Cd concentration in the intestinal homeostasis and health status of fish.

Phytosphingosine inhibits cell proliferation by damaging DNA in human cell lines.

Harmful cyanobacterial blooms have caused numerous biosecurity incidents owing to the production of hazardous secondary metabolites such as microcystin. Additionally, cyanobacteria also release many other components that have not been explored. We identified compounds of a toxic mixture exudated from a dominant, blooming species, Microcystis aeruginosa, and found that phytosphingosine (PHS) was one of the bioactive components. Since PHS exhibited toxicity and is deemed a hazardous substance by the European Chemicals Agency, we hypothesized that PHS is a potentially toxic compound in M. aeruginosa exudates. However, the mechanisms of PHS ecotoxicity remain unclear. We assessed the cytotoxicity of PHS using an in vitro cell model in eight human cell lines and observed that the nasopharyngeal carcinoma cell line CNE2 was the most sensitive. We exposed CNE2 cells to 0-25 µmol/L PHS for 24 hr to explore its toxicity and mechanism. PHS exposure resulted in abnormal nuclear morphology, micronuclei, and DNA damage. Moreover, PHS significantly inhibited cell proliferation and arrested cell cycle at S phase. The results of Western blot suggested that PHS increased the expression of DNA damage-related proteins (ATM, p-P53 and P21) and decreased the expression of S phase-related proteins (CDK2, CyclinA2 and CyclinE1), indicating the toxicological mechanism of PHS on CNE2 cells. These data provide evidence that PHS has genetic toxicity and inhibits cell proliferation by damaging DNA. Our study provides evidence that PHS inhibits cell proliferation by damaging DNA. While additional work is required, we propose that PHS been considered as a potentially toxic component in MaE in addition to other well-characterized secondary compounds.

Phytosphingosine-induced cell apoptosis via a mitochondrially mediated pathway.

Cyanobacterial blooms, usually dominated by Microcystis aeruginosa, pose a serious threat to global freshwater ecosystems owing to their production and release of various harmful secondary metabolites. Detection of the chemicals in M. aeruginosa exudates using metabolomics technology revealed that phytosphingosine (PHS) was one of the most abundant compounds. However, its specific toxicological mechanism remained unclear. CNE-2 cells were selected to illustrate the cytotoxic mechanism of PHS, and it was determined to cause excessive production of reactive oxygen species and subsequently damage the mitochondrial structure. Mitochondrial membrane rupture led to matrix mitochondrial membrane potential disintegration, which induced Ca2+ overload and interrupted ATP synthesis. Furthermore, rupture of the mitochondrial membrane induced the opening of the permeability transition pore, which caused the release of proapoptotic factors into the cytoplasm and the expression of apoptosis-related proteins Bax, Bcl-2, cytochrome-c and cleaved caspase-3 in CNE-2 cells. These events, in turn, activated the mitochondrially mediated intrinsic apoptotic pathway. A mitochondrial repair mechanism, namely, PINK1/Parkin-mediated mitophagy, was then blocked, which further promoted apoptosis. Our findings suggest that more attention should be paid to the ecotoxicity of PHS, which is already listed as a contaminant of emerging concern.

Assessment of metal mobility in sediment, commercial fish accumulation and impact on human health risk in a large shallow plateau lake in southwest of China.

Sediment heavy metal pollution in the Dianchi Lake has been a long-term environmental problem of concern. This study investigated the lake sediment heavy metal contamination level, mobility, commercial fish metal accumulation and its impact on human health. The results show high As, Hg and Cd concentration in the sediment, while Pb and Cr contamination are insignificant. Sediment sequential extraction analysis shows that Hg in sediment has the highest portion of mobile fraction, followed by As, while the portion of mobile fractions of Cd, Pb and Cr in sediment is very low. The high concentrations of Hg and As in surface water and porewater were consistent with the chemical fraction composition of the two elements in sediment. Three major commercial fish species, Culterichthys erythropterus, Carassius auratus and Hypophthalmichthys molitrix, were collected for analysis of metal concentrations in their muscles. Among the same size of fish, C. auratus has the highest As concentration due to its bottom habitat and omnivorous feeding habits. On the other hand, C. erythropterus has the highest Hg concentration due to its relatively high trophic level position. The average THQ value of metals in fish tissue decrease in the order of As > Hg > Pb > Cd > Cr and the total THQ of average metal concentration in fish species decreased in the order of C. auratus > C. erythropterus > H. molitrix. Both THQ and total THQ is below 1, suggested no non-carcinogenic human health risk of fish consumption. However, TR of As in C. auratus was above 1.00E-04 threshold value, indicated potential carcinogenic human health risk. The results from this study indicate that although moderately to heavily contamination of Hg, As, and Cd occurred in Dianchi Lake sediment, only Hg and As tend to transport to surface water and accumulate in commercial fish due to their higher mobility in sediment.

Blooming cyanobacteria alter water flea reproduction via exudates of estrogen analogues.

Cyanobacteria blooms are increasing globally, with further increases predicted in association with climate change. Recently, some cyanobacteria species have been identified as a source of estrogenic effects in aquatic animals. To explore possible estrogenic effects of Microcystis aeruginosa (an often-dominant cyanobacteria species) on zooplankton, we examined effects of cyanobacteria exudates (MaE, 2 × 104 and 4 × 105 cells/ml) on reproduction in Daphnia magna. We analyzed physiological, biochemical and molecular characteristics of exposed Daphnia via both chronic and acute exposures. MaE at both low and high cell density enhanced egg number (15.4% and 23.3%, respectively) and reproduction (37.7% and 52.4%, respectively) in D. magna similar to 10 µg/L estradiol exposure. In addition, both MaE of low and high cell densities increased population growth rate (15.8% and 19.6%, respectively) and reproductive potential (60% and 83%, respectively) of D. magna. These exudates promoted D. magna reproduction by stimulating 17ß-hydroxysteroid-dehydrogenase (17ß-HSD) activity and production of ecdysone and juvenile hormone, and by enhancing vitellogenin biosynthesis via up-regulating expression of Vtg1 and Vtg2. However, increased expression (6.6 times higher than controls) of a detoxification gene (CYP360A8) indicated that MaE might also induce toxicity in D. magna. Reproductive interference of zooplankton by blooming cyanobacteria might negatively affect foodwebs because MaE-induced zooplankton population increase would enhance grazing and reduce abundance of edible algae, thereby adding to the list of known disruptive properties of cyanobacterial blooms.