The development of saline-alkali lands has contributed to the increasing discharge of alkaline salt-laden wastewater, which poses a threat to aquatic organisms. However, the comprehensive effect of alkaline salt on Microcystis aeruginosa, a harmful cyanobacterium, remains unclear. In this study, the growth, physiology, cell ultrastructure and production of microcystin-LR (MC-LR) in Microcystis aeruginosa exposed to four levels of alkaline salt stress were evaluated. The growth of Microcystis aeruginosa was stimulated at an electrical conductivity (EC) of 2.5 mS/cm compared to the control, as supported by the increased cell density, photosynthetic pigment and protein contents. Microcystis aeruginosa could tolerate a certain level of alkaline salt (i.e., EC of 5 mS/cm) via increasing photosynthetic pigment contents to protect cells from alkaline salt stress, but the antioxidant defence system and cell ultrastructure were not affected. When EC increased to 7.5 mS/cm, alkaline salt caused oxidative stress and toxicity in Microcystis aeruginosa, as evidenced by analysis of the integrated biomarker response (IBR). Furthermore, the photosynthetic pigment and protein contents decreased, and cell apoptosis associated with ultrastructural changes was observed. Therefore, we propose that EC of 7.5 mS/cm is a threshold for growth of Microcystis aeruginosa. Additionally, the intracellular MC-LR content was stimulated by alkaline salt, and the highest value was observed at EC of 2.5 mS/cm. The extracellular MC-LR content increased with the increasing alkaline salt concentration. When EC was 7.5 mS/cm, the extracellular MC-LR content was significantly higher than in the control and was associated with the upregulated mcyH gene. This study recommends that more attention should be paid to the risk of Microcystis aeruginosa bloom and microcystin-LR pollution in lakes located in salinization regions.

Microcystin-LR (MC-LR) and glyphosate (GLY) have been classified as a Group 2B and Group 2A carcinogens for humans, respectively, and frequently found in aquatic ecosystems. However, data on the potential hazard of MC-LR and GLY exposure to the fish gut are relatively scarce. In the current study, a subacute toxicity test of zebrafish exposed to MC-LR (35 μg L⁻¹) and GLY (3.5 mg L⁻¹), either alone or in combination was performed for 21 d. The results showed that MC-LR or/and GLY treatment reduced the mRNA levels of tight junction genes (claudin-5, occludin, and zonula occludens-1) and altered the levels of diamine oxidase and D-lactic, indicating increased intestinal permeability in zebrafish. Furthermore, MC-LR and/or GLY treatment remarkably increased the levels of intestinal IL-1β and IL-8 but decreased the levels of IL-10 and TGF-β, indicating that MC-LR and/or GLY exposure induced an inflammatory response in the fish gut. MC-LR and/or GLY exposure also activated superoxide dismutase and catalase, generally upregulated the levels of p53, bax, bcl-2, caspase-3, and caspase-9, downregulated the levels of caspase-8 and caused notable histological injury in the fish gut. Moreover, MC-LR and/or GLY exposure also significantly altered the microbial community in the zebrafish gut and the expression of miRNAs (miR-146a, miR-155, miR-16, miR-21, and miR-223). Chronic exposure to MC-LR and/or GLY can induce intestinal damage in zebrafish, and this study is the first to demonstrate an altered gut microbiome and miRNAs in the zebrafish gut after MC-LR and GLY exposure.

The placenta is essential for sustaining the growth of the fetus. The aim of this study was to investigate the role of the placenta in MCLR-induced significant reduction in fetal weight, especially the changes in placental structure and function. Pregnant mice were intraperitoneally injected with MCLR (5 or 20 μg/kg) from gestational day (GD) 13 to GD17. The results showed MCLR reduced fetal weight and placenta weight. The histological specimens of the placentas were taken for light and electron microscopy studies. The internal space of blood vessels decreased obviously in the placental labyrinth layer of mice treated with MCLR. After the ultrastructural examination, the edema and intracytoplasmic vacuolization, dilation of the endoplasmic reticulum and corrugation of the nucleus were observed. In addition, maternal MCLR exposure caused a reduction of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) expression in placentae, a critical regulator of fetal development. Several genes of placental growth factors, such as Vegfα and Pgf and several genes of nutrient transport pumps, such as Glut1 and Pcft were depressed in placentas of MCLR-treated mice, however nutrient transporters Fatp1 and Snat4 were promoted. Moreover, significant increases in malondialdehyde (MDA) revealed the occurrence of oxidative stress caused by MCLR, which was also verified by remarkable decrease in the glutathione levels, total antioxidant capacity (T-AOC) as well as the activity of antioxidant enzymes. Real-time PCR and western blot analysis revealed that GRP78, CHOP, XBP-1, peIF2α and pIRE1 were remarkable increased in placentas of MCLR-treated mice, indicating that endoplasmic reticulum (ER) stress pathway was activated by MCLR. Furthermore, oxidative stress and ER stress consequently triggered apoptosis which contributed to the impairment of placental development. Collectively, these results suggest maternal MCLR exposure results in reduced fetal body weight, which might be associated with ROS-mediated endoplasmic reticulum stress and impairment in placental structure and function.

Microcystin-leucine-arginine (MC-LR) can cause male reproductive disorder. However, the underlying mechanism are not yet entirely elucidated. In this study, we aimed to investigated the effects of MC-LR on the integrity of blood-testis barrier (BTB) and the related molecular mechanisms. Both in vivo and in vitro experiments revealed that MC-LR caused disruption of BTB and gap junctions between Sertoli cells respectively, which was paralleled by the alteration of connexin43 (Cx43). Our data demonstrated that MC-LR decreased gap junction intercellular communication (GJIC) and impaired Cx43 expression by activating the phosphatidylinositol 3-kinase/Akt cascades. In addition, a possible protective effect of Icariin (ICA), a flavonoid isolated from Chinese medicinal herb, against MC-LR toxicity was investigated. The ICA prevented the degradation of GJIC and impairment of Cx43 induced by MC-LR via suppressing the Akt pathway. Together, our results confirmed that the expression of Cx43 induced by MC-LR was regulated in vivo and in vitro, which was involved in the destruction of BTB. Additionally, ICA seems to be able to mitigate the MC-LR toxic effects.

Microcystin (MC) elimination is a global challenge that is necessary for the health of humans and ecosystems. Biodegradation of MC, one of the most environmental-friendly methods, had previously been focused on the aerobic condition. In this study, two enrichment cultures from Taihu sediments possessed high capacity for MC-leucine arginine (MC-LR) anaerobic biodegradation. Meanwhile, it was firstly found that MC-LR underwent similar degradation process under anaerobic conditions to that in aerobic condition. Furthermore, a novel degradation pathway, hydrolyzing of Ala-Mdha to form a new linear MC-LR intermediate, was proposed under anaerobic conditions. Combining MC-LR degradation with microbial community analysis, this study deduced that Candidatus Cloacamonas acidaminovorans str. Evry may play an important role in the degradation of MC-LR. These findings suggest an additional pathway involved in the environmental cycle of MC-LR, which implies that the biotransformation of MC-LR might play an important role in eliminating MC-LR in eutrophic lake sediments under anaerobic conditions.

Our previous study showed that the tea extract, epigallocatechin-3-gallate (EGCG), protects against microcystin-LR (MC-LR) -mediated apoptosis of human umbilical vein endothelial cells (HUVECs); however, the mechanism underlying MC-LR-induced HUVEC apoptosis remains incompletely understood. In this study, we investigated whether the nuclear factor erythroid-like 2 (NRF2)/heme oxygenase-1 (HO-1) pathway, which regulates antioxidant transcriptional regulation of oxidative stress and apoptosis, is involved in this process. Mitochondrial membrane potential (MMP) and caspase-3/-9 activities were evaluated in HUVECs by JC-1 staining and colorimetric activity assay, and a DCFH-DA fluorescent probe assay was used to quantitate reactive oxygen species (ROS) generation. The effects of MC-LR, EGCG, NF2, and HO-1 on HUVEC apoptosis were explored by western blotting and small interfering RNA (siRNA) analyses. MC-LR treatment downregulated HUVEC mitochondrial membrane potential, and decreased levels of cytochrome c release and activated caspase-3/-9, ROS generation, consequently inducing HUVEC apoptosis. EGCG treatment attenuated MC-LR-mediated HUVEC oxidative stress and mitochondria-related apoptosis. EGCG induced NRF2/HO-1 expression and activation in MC-LR treated HUVECs, while downregulation of NRF2/HO-1 by specific siRNAs revealed that NRF2/HO-1 signaling was involved in EGCG attenuation of MC-LR-induced HUVEC apoptosis. Our findings indicate that EGCG treatment protects against MC-LR-mediated HUVEC apoptosis via activation of NRF2/HO-1 signaling.

Osteoporosis or enhanced bone loss is one of the most commonly occurring bone conditions in the world, responsible for higher incidence of fractures leading to increased morbidity and mortality in adults. Bone loss is affected by various environmental factors including diet, age, drugs, toxins etc. Microcystins are toxins produced by cyanobacteria with microcystin-LR being the most abundantly found around the world effecting both human and animal health. The present study demonstrates that MC-LR treatment induces bone loss and impairs both trabecular and cortical bone microarchitecture along with decreasing the mineral density and heterogeneity of bones in mice. This effect of MC-LR was found due to its immunomodulatory effects on the host immune system, wherein MC-LR skews both T cell (CD4+ and CD8+ T cells) and B cell populations in various lymphoid tissues. MC-LR further was found to significantly enhance the levels of osteoclastogenic cytokines (IL-6, IL-17 and TNF-α) along with simultaneously decreasing the levels of anti-osteoclastogenic cytokines (IL-10 and IFN-γ). Taken together, our study for the first time establishes a direct link between MC-LR intake and enhanced bone loss thereby giving a strong impetus to the naïve field of “osteo-toxicology”, to delineate the effects of various toxins (including cyanotoxins) on bone health.

Microcystin-LR (MC-LR) is the most abundant toxicant among microcystin variants produced by cyanobacteria. MC-induced toxicity is broadly reported to pose a threat to aquatic animals and humans and has been associated with the dysfunction of some organs such as liver and kidney. However, MC-induced neurotoxicity has not been well characterized after long-term exposure. This study was designed to investigate the neurotoxic effects after chronic oral administration of MC-LR. In our trial, C57/BL6 mice received MC-LR at 0, 1, 5, 10, 20 and 40 μg/L in drinking water for twelve months. Our data demonstrated that mitochondrial DNA (mtDNA) damage was evident in the damaged neurons as a result of chronic exposure. Histopathological abnormalities and mtDNA damage were observed in the hippocampus and cerebral cortex. Furthermore, MC-LR exerted distinct effects on these two brain regions. The hippocampus was more susceptible to the treatment of MC-LR compared with the cerebral cortex. However, no strong relationships were observed between the genotoxic effects and exposure doses. In conclusion, this study has provided a mtDNA-related mechanism for underlying chronic neurotoxicity of MC-LR and suggested the presence of differential toxicant effects on the hippocampus and cerebral cortex.

Microcystin-LR (MCLR) has been reported to cause developmental neurotoxicity in zebrafish, but there are few studies on the mechanisms of MCLR-induced transgenerational effects of developmental neurotoxicity. In this study, zebrafish were exposed to 0, 1, 5, and 25 μg/L MCLR for 60 days. The F1 zebrafish embryos from the above-mentioned parents were collected and incubated in clean water for 120 h for hatching. After examining the parental zebrafish and F1 embryos, MCLR was detected in the gonad of adults and F1 embryos, indicating MCLR could potentially be transferred from parents to offspring. The larvae also showed a serious hypoactivity. The contents of dopamine, dihydroxyphenylacetic acid (DOPAC), serotonin, gamma-aminobutyric acid (GABA) and acetylcholine (ACh) were further detected, but only the first three neurotransmitters showed significant reduction in the 5 and 25 μg/L MCLR parental exposure groups. In addition, the acetylcholinesterase (AChE) activity was remarkably decreased in MCLR parental exposure groups, while the expression levels of manf, bdnf, ache, htr1ab, htr1b, htr2a, htr1aa, htr5a, DAT, TH1 and TH2 genes coincided with the decreased content of neurotransmitters (dopamine, DOPAC and serotonin) and the activity of AChE. Neuronal development related genes, α1-tubulin, syn2a, mbp, gfap, elavl3, shha and gap43 were also measured, but gap43 was the gene only up-regulated. Our results demonstrated MCLR could be transferred to offspring, and subsequently induce developmental neurotoxicity in F1 zebrafish larvae by disturbing the neurotransmitter systems and neuronal development.

Microcystins (MCs) widely distributed in freshwaters have posed a significant risk to human health. Previous studies have demonstrated that exposure to MC-LR impairs pancreatic islet function, however, the underlying mechanisms still remain unclear. In the present study, we explored the role of endoplasmic reticulum (ER) impairment in β-cell dysfunction caused by MC-LR. The result showed that MC-LR modified ER morphology evidenced by increased ER amount and size at low doses (15, 30 or 60 μM) and vacuolar and dilated ER ultrastructure at high doses (100 or 200 μM). Also, insulin content showed increased at 15 or 30 μM but declined at 60, 100, or 200 μM, which was highly accordant with ER morphological alteration. Transcriptomic analysis identified a number of factors and several pathways associated with ER protein processing, ER stress, apoptosis, and diabetes mellitus in the cells treated with MC-LR compared with non-treated cells. Furthermore, MC-LR-induced ER stress significantly promoted the expression of PERK/eIF2α and their downstream targets (ATF4, CHOP, and Gadd34), which indicates that PERK-eIF2α-ATF4 pathway is involved in MC-LR-induced insulin deficiency. These results suggest that ER impairment is an important contributor to MC-LR-caused β-cell failure and provide a new insight into the association between MCs contamination and the occurrence of human diseases.