Microcystin-LR Induces Estrogenic Effects at Environmentally Relevant Concentration in Black-Spotted Pond Frogs (Pelophylax nigromaculatus): In Situ, In Vivo, In Vitro, and In Silico Investigations.

Harmful cyanobacterial blooms are frequent and intense worldwide, creating hazards for aquatic biodiversity. The potential estrogen-like effect of Microcystin-LR (MC-LR) is a growing concern. In this study, we assessed the estrogenic potency of MC-LR in black-spotted frogs through combined field and laboratory approaches. In 13 bloom areas of Zhejiang province, China, the MC-LR concentrations in water ranged from 0.87 to 8.77 µg/L and were correlated with sex hormone profiles in frogs, suggesting possible estrogenic activity of MC-LR. Tadpoles exposed to 1 µg/L, an environmentally relevant concentration, displayed a female-biased sex ratio relative to controls. Transcriptomic results revealed that MC-LR induces numerous and complex effects on gene expression across multiple endocrine axes. In addition, exposure of male adults significantly increased the estradiol (E2)/testosterone (T) ratio by 3.5-fold relative to controls. Downregulation of genes related to male reproductive endocrine function was also identified. We also showed how MC-LR enhances the expression of specific estrogen receptor (ER) proteins, which induce estrogenic effects by activating the ER pathway and hypothalamic-pituitary-gonadal (HPG) axis. In aggregate, our results reveal multiple lines of evidence demonstrating that, for amphibians, MC-LR is an estrogenic endocrine disruptor at environmentally relevant concentrations. The data presented here support the need for a shift in the MC-LR risk assessment. While hepatoxicity has historically been the focus of MC-LR risk assessments, our data clearly demonstrate that estrogenicity is a major mode of toxicity at environmental levels and that estrogenic effects should be considered for risk assessments on MC-LR going forward.

Impact of butylparaben on growth dynamics and microcystin-LR production in Microcystis aeruginosa.

The presence of butylparaben (BP), a prevalent pharmaceutical and personal care product, in surface waters has raised concerns regarding its impact on aquatic ecosystems. Despite its frequent detection, the toxicity of BP to the cyanobacterium Microcystis aeruginosa remains poorly understood. This study investigates the influence of BP on the growth and physiological responses of M. aeruginosa. Results indicate that low concentrations of BP (below 2.5 mg/L) have negligible effects on M. aeruginosa growth, whereas higher concentrations (5 mg/L and 10 mg/L) lead to significant growth inhibition. This inhibition is attributed to the severe disruption of photosynthesis, evidenced by decreased Fv/Fm values and chlorophyll a content. BP exposure also triggers the production of reactive oxygen species (ROS), resulting in elevated activity of antioxidant enzymes. Excessive ROS generation stimulates the production of microcystin-LR (MC-LR). Furthermore, lipid peroxidation and cell membrane damage indicate that high BP concentrations cause cell membrane rupture, facilitating the release of MC-LR into the environment. Transcriptome analysis reveals that BP disrupts energy metabolic processes, particularly affecting genes associated with photosynthesis, carbon fixation, electron transport, glycolysis, and the tricarboxylic acid cycle. These findings underscore the profound physiological impact of BP on M. aeruginosa and highlight its role in stimulating the production and release of MC-LR, thereby amplifying environmental risks in aquatic systems.

The effect and mechanism of combined exposure of MC-LR and NaNO2 on liver lipid metabolism.

Microcystin-LR (MC-LR) and sodium nitrite (NaNO2) co-exist in the environment and are hepatotoxic. The liver has the function of lipid metabolism, but the impacts and mechanisms of MC-LR and NaNO2 on liver lipid metabolism are unclear. Therefore, we established a chronic exposure model of Balb/c mice and used LO2 cells for in vitro verification to investigate the effects and mechanisms of liver lipid metabolism caused by MC-LR and NaNO2. The results showed that after 6 months of exposure to MC-LR and NaNO2, the lipid droplets content was increased, and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were raised in the liver (P < 0.05). Moreover, MC-LR and NaNO2 synergistically induced hepatic oxidative stress by decreasing total superoxide dismutase (T-SOD) activity and glutathione (GSH) levels and increasing malondialdehyde (MDA) content levels. In addition, the levels of Nrf2, HO-1, NQO1 and P-AMPK was decreased and Keap1 was increased in the Nrf2/HO-1 pathway. The key factors of lipid metabolism, SREBP-1c, FASN and ACC, were up-regulated in the liver. More importantly, there was a combined effect on lipid deposition of MC-LR and NaNO2 co-exposure. In vitro experiments, MC-LR and NaNO2-induced lipid deposition and changes in lipid metabolism-related changes were mitigated after activation of the Nrf2/HO-1 signaling pathway by the Nrf2 activator tertiary butylhydroquinone (TBHQ). Additionally, TBHQ alleviated the rise of reactive oxygen species (ROS) in LO2 cells induced by MC-LR and NaNO2. Overall, our findings indicated that MC-LR and NaNO2 can cause abnormal liver lipid metabolism, and the combined effects were observed after MC-LR and NaNO2 co-exposure. The Nrf2/HO-1 signal pathway may be a potential target for prevention and control of liver toxicity caused by MC-LR and NaNO2.

Magnetic recyclable visible light-driven Bi2WO6/Fe3O4/RGO for photocatalytic degradation of Microcystin-LR: Mechanism, pathway, and influencing factors.

Photocatalysis was an attractive strategy that had potential to tackle the Microcystin-LR (MC-LR) contamination of aquatic ecosystems. Herein, magnetic photocatalyst Fe3O4/Bi2WO6/Reduced graphene oxide composites (Bi2WO6/Fe3O4/RGO) were employed to degrade MC-LR. The removal efficiency and kinetic constant of the optimized Bi2WO6/Fe3O4/RGO (Bi2WO6/Fe3O4-40%/RGO) was 1.8 and 2.3 times stronger than the pure Bi2WO6. The improved activity of Bi2WO6/Fe3O4-40%/RGO was corresponded to the expanded visible light adsorption ability and reduction of photogenerated carrier recombination efficiency through the integration of Bi2WO6 and Fe3O4-40%/RGO. The MC-LR removal efficiency exhibited a positive tendency to the initial density of algae cells, fulvic acid, and the concentration of MC-LR decreased. The existed anions (Cl-, CO3-2, NO3-, H2PO4-) reduced MC-LR removal efficiency of Bi2WO6/Fe3O4-40%/RGO. The Bi2WO6/Fe3O4-40%/RGO could degrade 79.3% of MC-LR at pH = 7 after 180 min reaction process. The trapping experiments and ESR tests confirmed that the h+, ∙OH, and ∙O2- played a significant role in MC-LR degradation. The LC-MS/MS result revealed the intermediates and possible degradation pathways.

Co-exposure of microcystin-LR and nitrite induced kidney injury through TLR4/NLRP3/GSDMD-mediated pyroptosis.

The degradation of cyanobacterial blooms releases hazardous contaminants such as microcystin-LR (MC-LR) and nitrite, which may collectively exert toxicity on various bodily systems. To evaluate their individual and combined toxicity in the kidney, mice were subjected to different concentrations of MC-LR and/or nitrite over a 6-month period in this study. The results revealed that combined exposure to MC-LR and nitrite exacerbated renal pathological alterations and dysfunction compared to exposure to either compound alone. Specifically, the protein and mRNA expression of kidney injury biomarkers, such as kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), were notably increased in combined exposure group. Concurrently, co-exposure to MC-LR and nitrite remarkedly upregulated levels of proinflammatory cytokines TNF-α, IL-6 and IL-1ß, while decreasing the anti-inflammatory cytokine IL-10. Notably, MC-LR and nitrite exhibited synergistic effects on the upregulation of renal IL-1ß levels. Moreover, MC-LR combined with nitrite not only elevated mRNA levels of proinflammatory cytokines but also increased protein levels of pyroptosis biomarkers such as IL-1ß, Gasdermin D (GSDMD), and Cleaved-GSDMD. Mechanistic investigations revealed that co-exposure to MC-LR and nitrite promoted pyroptosis both in vivo and in vitro, possibly through the activation of the TLR4/NLRP3/GSDMD pathway. Pretreatment with TLR4 inhibitor and NLRP3 inhibitor effectively suppressed pyroptosis induced by the co-exposure of these two toxins in HEK293T cells. These findings provide compelling evidence that MC-LR combined with nitrite synergistically induces pyroptosis in the kidney by activating the TLR4/NLRP3/GSDMD pathway. Overall, this study significantly enhances our comprehension of how environmental toxins interact and induce harm to the kidneys, offering promising avenues for identifying therapeutic targets to alleviate their toxic effects on renal health.

Exposure to microcystin-LR promotes the progression of colitis-associated colorectal cancer by inducing barrier disruption and gut microbiota dysbiosis.

Microcystins (MCs) are secondary metabolites generated by cyanobacterial blooms, among which microcystin-LR (MC-LR) stands out as the most widely distributed variant in aquatic environments. However, the effects of MC-LR on the colorectum and its role in promoting colorectal tumor progression remain unclear. Therefore, this study aims to scrutinize the impact of MC-LR on a mice model of colitis-associated colorectal cancer and elucidate the potential underlying molecular mechanisms. In this study, we used AOM/DSS mice and orally administered MC-LR at doses of 40 µg/kg or 200 µg/kg. Exposure to MC-LR increased tumor burden, promoted tumor growth, shortened colon size, and decreased goblet cell numbers and tight junction protein levels in intestinal tissues. Additionally, exposure to MC-LR induced alterations in the structure of gut microbiota in the mouse colon, characterized by an increase in the relative abundance of Escherichia_coli and Shigella_sonnei, and a decline in the relative abundance of Akkermansia_muciniphila. Transcriptomic analysis revealed that MC-LR exposure activated the IL-17 signaling pathway in mouse colorectal tissues and participated in inflammation regulation and immune response. Immunofluorescence results demonstrated an increase in T-helper 17 (Th17) cell levels in mouse colorectal tumors following MC-LR exposure. The results from RT-qPCR revealed that MC-LR induced the upregulation of IL-6, IL-1ß, IL-10, IL-17A, TNF-α, CXCL1, CXCL2, CXCL5 and CCL20. The novelty of this study lies in its comprehensive approach to understanding the mechanisms by which MC-LR may contribute to CRC progression, offering new perspectives and valuable reference points for establishing guidance standards regarding MC-LR in drinking water. Our findings suggest that even at guideline value, MC-LR can have profound effects on susceptible mice, emphasizing the need for a reevaluation of guideline value and a deeper understanding of the role of environmental toxins in cancer progression.

Pubertal exposure to Microcystin-LR arrests spermatogonia proliferation by inducing DSB and inhibiting SIRT6 dependent DNA repair in vivo and in vitro.

The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2 µg/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.

Photoelectrochemical aptasensing with methylene blue filled Ni-MOFs nanocomposite by spatial confinement for microcystin-LR detection.

Microcystin LR (MC-LR) is a hazardous cyanotoxin produced by cyanobacteria during freshwater eutrophication, which can cause liver cancer. Here, a photoelectrochemical (PEC) aptasensor based on methylene blue (MB)-loaded Ni-MOF composite (Ni-MOF/MB) with spatial confinement was constructed for the sensitive detection of MC-LR. Ni-MOF with two-dimensional sheet structure was prepared via a liquid-liquid interface synthesis method with environmental-friendly solvent and milder reaction conditions. Benefiting from the uniform pore size, Ni-MOF acted as reaction platform to anchor the photosensitive molecule MB. The electron donor, ascorbic acid (AA), was produced by alkaline phosphatase (ALP) loaded on DNA strand catalyzing ascorbic acid phosphate. The generated AA was absorbed by Ni-MOF/MB, thereby effectively improving the utilization of AA and avoiding the external environment interferences to enlarge the photocurrent of MB. For analysis, ALP-labeled aptamer can specifically recognize MC-LR by forming a complex to strip from aptasensor, thus leading to a  decreased photocurrent. The developed PEC aptasensor offered a linear range of 10 fM-100 pM with a detection limit of 6 fM. It was successfully employed for detecting MC-LR in farm water and fish meat, and the results were validated by ultrahigh-performance liquid chromatography-mass spectrometry. This method presents a new idea of MOF-limited domain for PEC aptasensing.

Mutations at Two Key Sites in PP2A Safeguard Caenorhabditis elegans Neurons from Microcystin-LR Toxicity.

Microcystin-LR (MC-LR) is a secondary metabolite produced by cyanobacteria, globally renowned for its potent hepatotoxicity. However, an increasing body of research suggests that it also exhibits pronounced neurotoxicity. PP2A is a fundamental intracellular phosphatase that plays a pivotal role in cell development and survival. Although extensive research has focused on the binding of MC-LR to the C subunit of PP2A, few studies have explored the key amino acid sites that can prevent the binding of MC-LR to PP2A-C. Due to the advantages of Caenorhabditis elegans (C. elegans), such as ease of genetic editing and a short lifespan, we exposed nematodes to MC-LR in a manner that simulated natural exposure conditions based on MC-LR concentrations in natural water bodies (immersion exposure). Our findings demonstrate that MC-LR exerts comprehensive toxicity on nematodes, including reducing lifespan, impairing reproductive capabilities, and diminishing sensory functions. Notably, and for the first time, we observed that MC-LR neurotoxic effects can persist up to the F3 generation, highlighting the significant threat that MC-LR poses to biological populations in natural environments. Furthermore, we identified two amino acid sites (L252 and C278) in PP2A-C through mutations that prevented MC-LR binding without affecting PP2A activity. This discovery was robustly validated through behavioral studies and neuronal calcium imaging using nematodes. In conclusion, we identified two crucial amino acid sites that could prevent MC-LR from binding to PP2A-C, which holds great significance for the future development of MC-LR detoxification drugs.

Degradation performance and mechanism of microcystins in aquaculture water using low-temperature plasma technology.

The eutrophication of aquaculture water bodies seriously restricts the healthy development of the aquaculture industry. Among them, microcystins are particularly harmful. Therefore, the development of technologies for degrading microcystins is of great significance for maintaining the healthy development of the aquaculture industry. The feasibility and mechanism of removing microcystins-LR by dielectric barrier discharge (DBD) plasma were studied. DBD discharge power of 49.6 W and a treatment time of 40 min were selected as the more suitable DBD parameters, resulting in microcystin-LR removal efficiency of 90.4%. Meanwhile, the effects of initial microcystin-LR concentration, initial pH value, turbidity, anions on the degradation effect of microcystin-LR were investigated. The removal efficiency of microcystin-LR decreased with the increase of initial microcystin-LR concentration and turbidity. The degradation efficiency of microcystin-LR at pH 4.5 and 6.5 is significantly higher than that at pH 8.5 and 3.5. HCO3- can inhibit the removal efficiency of microcystin-LR. Furthermore, five intermediates products (m/z = 1029.5, 835.3, 829.3, 815.4, 642.1) were identified in this study, and the toxicity analysis of these degradation intermediates indicated that DBD treatment can reduce the toxicity of microcystin-LR. e－aq, •OH, H2O2, and O3 have been shown to play a major role in the degradation of microcystin-LR, and the contribution ranking of these active species is e－aq > â€¢OH > H2O2 > O3. The application of DBD plasma technology in microcystin-LR removal and detoxification has certain development potential.

Microcystin-LR and polystyrene microplastics jointly lead to hepatic histopathological damage and antioxidant dysfunction in male zebrafish.

The co-occurrence of cyanobacterial blooms and nano-microplastic pollution in the water is becoming an emerging risk. To assess the combined hepatotoxicity of microcystin-LR (MC-LR) and polystyrene microplastics (PSMPs) on zebrafish (Danio rerio), male adult zebrafish were exposed to single MC-LR (0, 1, 5, 25 µg/L) and a mixture of MC-LR and PSMPs (100 µg/L). After 60 d exposure, the results indicated that PSMPs significantly increased the MC-LR bioaccumulation in the livers in contrast to the single 25 µg/L MC-LR treatment group. Moreover, the severity of hepatic pathological lesions was aggravated in the MC-LR + PSMPs treatment groups, which were mainly characterized by cellular vacuolar degeneration, swollen hepatocytes, and pyknotic nucleus. The ultrastructural changes also proved that PSMPs combined with MC-LR could enhance the swollen mitochondria and dilated endoplasmic reticulum. The biochemical results, including increased malondialdehyde (MDA) and decreased glutathione (GSH), indicated that PSMPs intensified the MC-LR-induced oxidative damage in the combined treatment groups. Concurrently, alterations of sod1 and keap1a mRNA levels also confirmed that PSMPs together with MC-LR jointly lead to enhanced oxidative injury. Our findings demonstrated that PSMPs enhanced the MC-LR bioavailability by acting as a vector and exacerbating the hepatic injuries and antioxidant dysfunction in zebrafish.

Transgenerational effects of extracts containing Microcystin-LR exposure on reproductive toxicity and offspring growth inhibition in a model organism zebrafish.

Cyanobacteria cell lysates release numerous toxic substances (e.g., cyanotoxins) into the water, posing a serious threat to human health and aquatic ecosystems. Microcystins (MCs) are among the most abundant cyanotoxins in the cell lysates, with microcystin-LR (MC-LR) being one of the most common and highly toxic congeners. In this study, zebrafish (Danio rerio) were exposed to different levels MC-LR that from extracts of Microcystis aeruginosa. Changes in the MC-LR accumulations, organ coefficients, and antioxidant enzyme activities in the zebrafish were analyzed. Transgenerational reproductive toxicity of MC-LR in the maternal and paternal generations was further investigated, as well as the influences of extracts containing MC-LR exposures of the F1 on the growth of zebrafish. The study found that high levels of MC-LR could be detected in the major organs of adult zebrafish, particularly in spleen. Notably, concentration of MC-LR in the spermary was significantly higher than that in the ovarium. MC-LR could induce oxidative damage by affecting the activities of catalase and superoxide dismutase. Inherited from F0, MC-LR led to impaired development in the F1 generation. Difference in offspring survival rates could be observed in the groups with different MC-LR levels of maternal and paternal exposures. This study reveals transgenerational effects of MC-LR on the reproductive toxicity and offspring growth inhibition to the aquatic organisms, which should be emphasized in the future ecological risk assessment.

Toxic effects of nanoplastics and microcystin-LR coexposure on the liver-gut axis of Hypophthalmichthys molitrix.

Plastic products and nutrients are widely used in aquaculture facilities, resulting in copresence of nanoplastics (NPs) released from plastics and microcystins (MCs) from toxic cyanobacteria. The potential effects of NPs-MCs coexposure on aquatic products require investigation. This study investigated the toxic effects of polystyrene (PS) NPs and MC-LR on the gut-liver axis of silver carp Hypophthalmichthys molitrix, a representative commercial fish, and explored the effects of the coexposure on intestinal microorganism structure and liver metabolic function using traditional toxicology and multi-omics association analysis. The results showed that the PS-NPs and MC-LR coexposure significantly shortened villi length, and the higher the concentration of PS-NPs, the more obvious the villi shortening. The coexposure of high concentrations of PS-NPs and MC-LR increased the hepatocyte space in fish, and caused obvious loss of gill filaments. The diversity and richness of the fish gut microbes significantly increased after the PS-NPs exposure, and this trend was amplified in the copresence of MC-LR. In the coexposure, MC-LR contributed more to the alteration of fish liver metabolism, which affected the enrichment pathway in glycerophospholipid metabolism and folic acid biosynthesis, and there was a correlation between the differential glycerophospholipid metabolites and affected bacteria. These results suggested that the toxic mechanism of PS-NPs and MC-LR coexposure may be pathological changes of the liver, gut, and gill tissues, intestinal microbiota disturbance, and glycerophospholipid metabolism imbalance. The findings not only improve the understanding of environmental risks of NPs combined with other pollutants, but also provide potential microbiota and glycerophospholipid biomarkers in silver carp.

Metabolomics reveals the lipid metabolism disorder in Pelophylax nigromaculatus exposed to environmentally relevant levels of microcystin-LR.

Cyanobacterial blooms have emerged as a significant environmental issue worldwide in recent decades. However, the toxic effects of microcystin-LR (MC-LR) on aquatic organisms, such as frogs, have remained poorly understood. In this study, frogs (Pelophylax nigromaculatus) were exposed to environmentally relevant concentrations of MC-LR (0, 1, and 10 µg/L) for 21 days. Subsequently, we assessed the impact of MC-LR on the histomorphology of the frogs' livers and conducted a global MS-based nontarget metabolomics analysis, followed by the determination of substances involved in lipid metabolism. Results showed that MC-LR significantly induced histological alterations in the frogs' hepatopancreas. Over 200 differentially expressed metabolites were identified, primarily enriched in lipid metabolism. Biochemical analysis further confirmed that MC-LR exposure led to a disorder in lipid metabolism in the frogs. This study laid the groundwork for a mechanistic understanding of MC-LR toxicity in frogs and potentially other aquatic organisms.

Ultrastructural, molecular and haemato-immunological changes: Multifaceted toxicological effects of microcystin-LR in rohu, Labeo rohita.

No water body is resilient to afflicts of algal bloom, if goes unmanaged. With the increasing trend of intensification, eutrophication and climate change, Labeo rohita (rohu) is highly anticipated to suffer from the deleterious effects of bloom and eventually its toxins. A comprehensive study was conducted to understand the toxicopathological effects of microcystin-LR (MC-LR) in rohu following intraperitoneal injection of 96 h-LD50 dose i.e., 713 µg kg-1. Substantial changes in micro- and ultrastructural level were evident in histopathology and transmission electron microscope (TEM) study. The haematological, biochemical, cellular and humoral innate immune biomarkers were significantly altered (p < 0.05) in MC-LR treated fish. The mRNA transcript levels of IL-1ß, IL-10, IgM and IgZ in liver and kidney tissues were significantly up-regulated in 12 hpi and declined in 96 hpi MC-LR exposed fish. The relative mRNA expression of caspase 9 in the liver and kidney indicates mitochondrial-mediated apoptosis which was strongly supported by TEM study. In a nutshell, our study illustrates for the first time MC-LR induced toxicological implications in rohu displaying immunosuppression, enhanced oxidative stress, pathophysiology, modulation in mRNA transcription, genotoxicity, structural and ultrastructural alterations signifying it as a vulnerable species for MC-LR intoxication.

Microcystin-LR improves anti-tumor efficacy of oxaliplatin through induction of M1 macrophage polarization.

Tumor-associated macrophages within the tumor microenvironment play an immunosuppressive role by promoting tumor growth and immune evasion. Macrophages are highly plastic and can be stimulated to adopt an anti-tumor M1 phenotype. In this study, we used microcystin-LR (MC-LR), a cyclic heptapeptide produced by cyanobacteria, to induce in vitro macrophage innate immunity and transition into the anti-tumor M1 phenotype. MC-LR was also tested in vivo in a mouse model of colorectal cancer. An intraperitoneal injection of MC-LR increased the proportion of CD86⁺ M1 macrophages and triggered the maturation of CD11c⁺ dendritic cells within tumor tissues. MC-LR combined with the chemotherapeutic drug oxaliplatin significantly inhibited tumor growth in vivo. Flow cytometry analysis revealed increased infiltration of activated cytotoxic (CD8⁺, PD-1⁺) T-cells and anti-tumor cytokines (IFNγ and Granzyme B) in the tumor tissues of the combination therapy group, suggesting that this may be the primary mechanism behind the anti-tumor effect of the combination treatment. These findings indicate that MC-LR regulates the immune stimulation of macrophage polarization and dendritic cell maturation, effectively reversing tumor immunosuppression, activating an anti-tumor immune response, and enhancing tumor therapy.

The effects of nanoplastics and microcystin-LR coexposure on Aristichthys nobilis at the early developmental stages.

Nanoplastics (NPs) and microcystin-LR (MC-LR) are two common and harmful pollutants in water environments, especially at aquafarm where are full of plastic products and algae. It is of great significance to study the toxic effects and mechanisms of the NPs and/or MC-LR on fish at the early stage. In this study, the embryo and larvae of a filtering-feeding fish, Aristichthys nobilis, were used as the research objects. The results showed that the survival and hatching rates of the embryo were not significantly affected by the environmental concentration exposure of these two pollutants. Scanning electron microscopy (SEM) observation displayed that NPs adhered to the surface of the embryo membrane. Transcriptomic and bioinformatic analyses revealed that the NPs exposure activated neuromuscular junction development and skeletal muscle fiber in larvae, and affected C5-Branched dibasic acid metabolism. The metabolic and biosynthetic processes of zeaxanthin, xanthophyll, tetraterpenoid, and carotenoid were suppressed after the MC-LR exposure, which was harmful to the retinol metabolism of fish. Excessive production of superoxide dismutase (SOD) was detected under the MC-LR exposure. The MC-LR and NPs coexposure triggered primary immunodeficiency and adaptive immune response, leading to the possibility of reduced fitness of A.nobilis during the development. Collectively, our results indicate that environmental concentration NPs and MC-LR coexposure could cause toxic damage and enhance sick risk in A.nobilis, providing new insights into the risk of NPs and MC-LR on filtering-feeding fish.

Introducing fluorescent probe technology for detecting microcystin-LR in the water and cells.

BACKGROUND: For a long time, the environment hazards caused by cyanobacteria bloom and associated microcystins have attracted attention worldwide. Microcystin-LR (MC-LR) is the most widely distributed and most toxic toxin. At present, numerous MC-LR detection methods exist many drawbacks. Therefore, a quick and accurate method for identifying and detecting MC-LR is crucial and necessary. In this work, we strived to introduce a novel fluorescence assay to detect MC-LR in the water and cells. RESULTS: According to the special spatial configuration and physicochemical properties of MC-LR, we designed and constructed six fluorescent probes. The design concepts of the probes were exhaustively elaborated. MC-YdTPA, MC-YdTPE, MC-RdTPA, and MC-RdTPE could show significant fluorescence enhancement in MC-LR solution. Significantly, MC-YdTPA, MC-YdTPE, and MC-RdTPA could also response well in the cells treated with MC-LR, demonstrating these fluorescent probes' values. The recognition mechanism between probes and MC-LR were also deeply explored: (1) The polyphenylene ring structure of probes may have nested or hydrogen bond weak interaction with the ring structure of MC-LR. (2) The probes can generate a reaction to the hydrogen ions ionized by MC-LR. SIGNIFICANCE: We proposed the novel ideas for designing MC-LR probes. This research can provide valuable experiences and important assistance in synthesizing MC-LR fluorescent probes. We expect that this work may bring new ideas to develop fluorescent probes for researching MC-LR in vivo and in vitro.

Suspended particulate matter-biofilm aggregates benefit microcystin removal in turbulent water but trigger toxicity toward Daphnia magna.

Suspended particulate matter (SPM) and biofilm are critical in removing contaminants in aquatic environments, but the environmental behavior and ecological toxicity of SPM-biofilm aggregates modulated by turbulence intensities are largely unknown. This study determined the removal pathways of microcystin-LR (MC-LR) by SPM and its biofilm under different turbulence intensities (2.25 × 10-3, 1.01 × 10-2, and 1.80 × 10-2 m2/s3). Then, we evaluated the toxicity of SPM-biofilm aggregates to Daphnia magna. The results revealed that SPM contributed to the adsorption of MC-LR, and the removal of MC-LR can be accelerated with biofilm formation on SPM, with 95.66 % to 97.45 % reduction in MC-LR concentration under the studied turbulence intensities. Higher turbulence intensity triggered more frequent contact of SPM and MC-LR, formed compact but smaller clusters of SPM-biofilm aggregates, and enhanced the abundance of mlrA and mlrB; thus benefiting the adsorption, biosorption, and biodegradation of MC-LR. Furthermore, the SPM-biofilm aggregates formed in turbulent water triggered oxidative stress to Daphnia magna, while a weak lethal toxic effect was identified under moderate turbulence intensity. The results indicate that the toxicity of SPM-biofilm aggregates fail to display a linear relationship with turbulence intensity. These findings offer new perspectives on understanding the environmental behavior and ecological outcomes of SPM and its biofilms in turbulent aquatic environments.

Rapid and Easy Detection of Microcystin-LR Using a Bioactivated Multi-Walled Carbon Nanotube-Based Field-Effect Transistor Sensor.

In this study, we developed a multi-walled carbon nanotube (MWCNT)-based field-effect transistor (MWCNT-FET) sensor with high sensitivity and selectivity for microcystin-LR (MC-LR). Carboxylated MWCNTs were activated with an MC-LR-targeting aptamer (MCTA). Subsequently the bioactivated MWCNTs were immobilized between interdigitated drain (D) and source (S) electrodes through self-assembly. The top-gated MWCNT-FET sensor was configured by dropping the sample solution onto the D and S electrodes and immersing a Ag/AgCl electrode in the sample solution as a gate (G) electrode. We believe that the FET sensor's conduction path arises from the interplay between the MCTAs, with the applied gate potential modulating this path. Using standard instruments and a personal computer, the sensor's response was detected in real-time within a 10 min time frame. This label-free FET sensor demonstrated an impressive detection capability for MC-LR in the concentration range of 0.1-0.5 ng/mL, exhibiting a lower detection limit of 0.11 ng/mL. Additionally, the MWCNT-FET sensor displayed consistent reproducibility, a robust selectivity for MC-LR over its congeners, and minimal matrix interferences. Given these attributes, this easily mass-producible FET sensor is a promising tool for rapid, straightforward, and sensitive MC-LR detection in freshwater environments.