Imaging lutein and zeaxanthin in the human retina with confocal resonance Raman microscopy.

Lutein and zeaxanthin are xanthophyll carotenoids that are highly concentrated in the human macula, where they protect the eye from oxidative damage and improve visual performance. Distinguishing lutein from zeaxanthin in images of the human retina in vivo or in donor eye tissues has been challenging because no available technology has been able to reliably differentiate between these two carotenoids, which differ only in the position of one C = C bond. Here, we report the differential distributions of lutein and zeaxanthin in human donor retinas mapped with confocal resonance Raman microscopy. Zeaxanthin is highly concentrated in the fovea, extending from the inner to the outer limiting membranes, with especially high concentrations in the outer plexiform layer, while lutein is much more diffuse at relatively lower concentration. Our results imply that zeaxanthin may play a more important role than lutein in human macular health and disease.

Hawthorn fruit extract protect against MC-LR-induced hepatotoxicity by attenuating oxidative stress and apoptosis.

Microcystins (MCs) is a class of cyclic heptapeptide compounds with biological activity. There is no effective treatment for liver injury caused by MCs. Hawthorn is a medicinal and edible plant traditional Chinese medicine with hypolipidemic, reducing inflammation and oxidative stress in the liver. This study discussed the protective effect of hawthorn fruit extract (HFE) on liver damage caused by MC-LR and the underlying molecular mechanism. After MC-LR exposure, pathological changes were observed and hepatic activity of ALT, AST and ALP were increased obviously, but they were remarkably restored with HFE administration. In addition, MC-LR could significantly reduce SOD activity and increase MDA content. Importantly, MC-LR treatment resulted in mitochondrial membrane potential decreased, and Cytochrome C release, eventually leading to cell apoptosis rate increase. HFE pretreatment could significantly alleviate the above abnormal phenomena. To examine the mechanism of protection, the expression of critical molecules in the mitochondrial apoptosis pathway was examined. The levels of Bcl-2 was inhibited, and the levels of Bax, Caspase-9, Cleaved Caspase-9, and Cleaved caspase-3 were upregulated after MC-LR treatment. HFE reduced MC-LR-induced apoptosis via reversing the expression of key proteins and genes in the mitochondrial apoptotic pathway. Hence, HFE could alleviate MC-LR induced hepatotoxicity by reducing oxidative stress and apoptosis.

PI3K/AKT/mTOR pathway mediated-cell cycle dysregulation contribute to malignant proliferation of mouse spermatogonia induced by microcystin-leucine arginine.

Environmental cyanotoxin exposure may be a trigger of testicular cancer. Activation of PI3K/AKT/mTOR signaling pathway is the critical molecular event in testicular carcinogenesis. As a widespread cyanotoxin, microcystin-leucine arginine (MC-LR) is known to induce cell malignant transformation and tumorigenesis. However, the effects of MC-LR on the regulatory mechanism of PI3K/AKT/mTOR pathway in seminoma, the most common testicular tumor, are unknown. In this study, mouse spermatogonia cell line (GC-1) and nude mice were used to investigate the effects and mechanisms of MC-LR on the malignant transformation of spermatogonia by nude mouse tumorigenesis assay, cell migration invasion assay, western blot, and cell cycle assay, and so forth. The results showed that, after continuous exposure to environmentally relevant concentrations of MC-LR (20 nM) for 35 generations, the proliferation, migration, and invasion abilities of GC-1 cells were increased by 120%, 340%, and 370%, respectively. In nude mice, MC-LR-treated GC-1 cells formed tumors with significantly greater volume (0.998 ± 0.768 cm3 ) and weight (0.637 ± 0.406 g) than the control group (0.067 ± 0.039 cm3 ; 0.094 ± 0.087 g) (P < .05). Furthermore, PI3K inhibitor Wortmannin inhibited the PI3K/AKT/mTOR pathway and its downstream proteins (c-MYC, CDK4, CCND1, and MMP14) activated by MC-LR. Blocking PI3K alleviated MC-LR-induced cell cycle disorder and malignant proliferation, migration and invasive of GC-1 cells. Altogether, our findings suggest that MC-LR can induce malignant transformation of mouse spermatogonia, and the PI3K/AKT/mTOR pathway-mediated cell cycle dysregulation may be an important target for malignant proliferation. This study provides clues to further reveal the etiology and pathogenesis of seminoma.

HDL is the primary transporter for carotenoids from liver to retinal pigment epithelium in transgenic ApoA-I-/-/Bco2-/- mice.

Supplementation with antioxidant carotenoids is a therapeutic strategy to protect against age-related macular degeneration (AMD); however, the transport mechanism of carotenoids from the liver to the retina is still not fully understood. Here, we investigate if HDL serves as the primary transporter for the macular carotenoids. ApoA-I, the key apolipoprotein of HDL, was genetically deleted from BCO2 knockout (Bco2-/-) mice, a macular pigment mouse model capable of accumulating carotenoids in the retina. We then conducted a feeding experiment with a mixed carotenoid chow (lutein:zeaxanthin:ß-carotene = 1:1:1) for one month. HPLC data demonstrated that the total carotenoids were increased in the livers but decreased in the serum, retinal pigment epithelium (RPE)/choroids, and retinas of ApoA-I-/-/Bco2-/- mice compared to Bco2-/- mice. In detail, ApoA-I deficiency caused a significant increase of ß-carotene but not lutein and zeaxanthin in the liver, decreased all three carotenoids in the serum, blocked the majority of zeaxanthin and ß-carotene transport to the RPE/choroid, and dramatically reduced ß-carotene and zeaxanthin but not lutein in the retina. Furthermore, surface plasmon resonance spectroscopy (SPR) data showed that the binding affinity between ApoA-I and ß-carotene â‰« zeaxanthin > lutein. Our results show that carotenoids are transported from the liver to the eye mainly by HDL, and ApoA-I may be involved in the selective delivery of macular carotenoids to the RPE.

Long-term exposure to low concentrations of MC-LR induces blood-testis barrier damage through the RhoA/ROCK pathway.

Microcystin-leucine arginine (MC-LR), an emerging water pollutant, produced by cyanobacteria, has an acute testicular toxicity. However, little is known about the chronic toxic effects of MC-LR exposure on the testis at environmental concentrations and the underlying molecular mechanisms. In this study, C57BL/6 J mice were exposed to different low concentrations of MC-LR for 6, 9 and 12 months. The results showed that MC-LR could cause testis structure loss, cell abscission and blood-testis barrier (BTB) damage. Long-term exposure of MC-LR also activated RhoA/ROCK pathway, which was accompanied by the rearrangement of α-Tubulin. Furthermore, MC-LR reduced the levels of the adherens junction proteins (N-cadherin and ß-catenin) and the tight junction proteins (ZO-1 and Occludin) in a dose- and time-dependent way, causing BTB damage. MC-LR also reduced the expressions of Occludin, ZO-1, ß-catenin, and N-cadherin in TM4 cells, accompanied by a disruption of cytoskeletal proteins. More importantly, the RhoA inhibitor Rhosin ameliorated these MC-LR-induced changes. Together, these new findings suggest that long-term exposure to MC-LR induces BTB damage through RhoA/ROCK activation: involvement of tight junction and adherens junction changes and cytoskeleton disruption. This study highlights a new mechanism for MC-LR-induced BTB disruption and provides new insights into the cause and treatment of BTB disruption.

MicroRNA-122 overexpression promotes apoptosis and tumor suppressor gene expression induced by microcystin-leucine arginine in mouse liver.

Microcystin-leucine arginine (MC-LR), an important hepatoxin, has the effect of promoting hepatocarcinogenesis. MicroRNA-122 (miR-122), an important tumor suppressor in liver, plays an important role in promoting cell apoptosis. Previous studies found that the expression of miR-122 was reduced after MC-LR exposure in liver. In this study, C57BL/6 mice were exposed to saline, negative control agomir, and MC-LR with or without miR-122 agomir transfection. The results indicated that MC-LR promoted the expressions of tumor suppressor genes and decreased the expressions of anti-apoptotic proteins B cell lymphoma-2 (Bcl-2) and Bcl-2-like 2 (Bcl-w), causing hepatocyte apoptosis. Under MC-LR exposure, miR-122 agomir transfection could further increase the expressions of tumor suppressor genes and the release of cytochrome-c (Cyt-c) and decrease the expressions of Bcl-2 and Bcl-w. In conclusion, miR-122 reduction can mitigate MC-LR-induced apoptosis to a certain extent, which in turn, it is likely to have contributed to MC-LR-induced hepatocarcinogenesis.

Update on the adverse effects of microcystins on the liver.

Microcystins (MCs) are the most common cyanobacteria toxins in eutrophic water, which have strong hepatotoxicity. In the past decade, epidemiological and toxicological studies on liver damage caused by MCs have proliferated, and new mechanisms of hepatotoxicity induced by MCs have also been discovered and confirmed. However, there has not been a comprehensive and systematic review of these new findings. Therefore, this paper summarizes the latest advances in studies on the hepatotoxicity of MCs to reveal the effects and mechanisms of hepatotoxicity induced by MCs. Current epidemiological studies have confirmed that symptoms or signs of liver damage appear after human exposure to MCs, and a long time of exposure can even lead to liver cancer. Toxicological studies have shown that MCs can affect the expression of oncogenes by activating cell proliferation pathways such as MAPK and Akt, thereby promoting the occurrence and development of cancer. The latest evidence shows that epigenetic modifications may play an important role in MCs-induced liver cancer. MCs can cause damage to the liver by inducing hepatocyte death, mainly manifested as apoptosis and necrosis. The imbalance of liver metabolic homeostasis may be involved in hepatotoxicity induced by MCs. In addition, the combined toxicity of MCs and other toxins are also discussed in this article. This detailed information will be a valuable reference for further exploring of MCs-induced hepatotoxicity.

The latest advances in the reproductive toxicity of microcystin-LR.

Microcystin-LR (MC-LR) is an emerging environmental pollutant produced by cyanobacteria that poses a threat to wild life and human health. In recent years, the reproductive toxicity of MC-LR has gained widespread attention, a large number of toxicological studies have filled the gaps in past research and more molecular mechanisms have been elucidated. Hence, this paper reviews the latest research advances on MC-LR-induced reproductive toxicity. MC-LR can damage the structure and function of the testis, ovary, prostate, placenta and other organs of animals and then reduce their fertility. Meanwhile, MC-LR can also be transmitted through the placenta to the offspring causing trans-generational and developmental toxicity including death, malformation, growth retardation, and organ dysfunction in embryos and juveniles. The mechanisms of MC-LR-induced reproductive toxicity mainly include the inhibition of protein phosphatase 1/2 A (PP1/2 A) activity and the induction of oxidative stress. On the one hand, MC-LR triggers the hyperphosphorylation of certain proteins by inhibiting intracellular PP1/2 A activity, thereby activating multiple signaling pathways that cause inflammation and blood-testis barrier destruction, etc. On the other hand, MC-LR-induced oxidative stress can result in cell programmed death via the mitochondrial and endoplasmic reticulum pathways. It is worth noting that epigenetic modifications are also involved in reproductive cell apoptosis, which may be an important direction for future research. Furthermore, this paper proposes for the first time that MC-LR can produce estrogenic effects in animals as an environmental estrogen. New findings and suggestions in this review could be areas of interest for future research.

Microcystin-LR induces ovarian injury and apoptosis in mice via activating apoptosis signal-regulating kinase 1-mediated P38/JNK pathway.

As an emerging pollutant in the aquatic environment, microcystin-LR (MC-LR) can enter the body through multiple pathways, and then induce apoptosis and gonadal damage, affecting reproductive function. Previous studies focused on male reproductive toxicity induced by MC-LR neglecting its effects on females. The apoptotic signal-regulated kinase 1 (ASK1) is an upstream protein of P38/JNK pathway, closely associated with apoptosis and organ damage. However, the role of ASK1 in MC-LR-induced reproductive toxicity is unclear. Therefore, this study investigated the role of ASK1 in mouse ovarian injury and apoptosis induced by MC-LR. After MC-LR exposure, ASK1 expression in mouse ovarian granulosa cells was increased at the protein and mRNA levels, and decreased following pretreatment by antioxidant N-acetylcysteine, suggesting that MC-LR-induced oxidative stress has a regulatory role in ASK1 expression. Inhibition of ASK1 expression with siASK1 and NQDI-1 could effectively alleviate MC-LR-induced mitochondrial membrane potential damage and apoptosis in ovarian granulosa cells, as well as pathological damage, apoptosis and the decreased gonadal index in ovaries of C57BL/6 mice. Moreover, the P38/JNK pathway and downstream apoptosis-related proteins (P-P38, P-JNK, P-P53, Fas) and genes (MKK4, MKK3, Ddit3, Mef2c) were activated in vivo and vitro, but their activation was restrained after ASK1 inhibition. Data presented herein suggest that the ASK1-mediated P38/JNK pathway is involved in ovarian injury and apoptosis induced by MC-LR in mice. It is confirmed that ASK1 has an important role in MC-LR-induced ovarian injury, which provides new insights for preventing MCs-induced reproductive toxicity in females.

The activated ATM/p53 pathway promotes autophagy in response to oxidative stress-mediated DNA damage induced by Microcystin-LR in male germ cells.

Microcystin-LR (MC-LR) is an intracellular toxin with multi-organ toxicity and the testis is one of its important target organs. Although there is increasing research on MC-LR in male reproductive toxicity, the association between DNA damage and autophagy induced by MC-LR in male germ cells are still unclear. Therefore, it is important to explore the mechanism of MC-LR-induced DNA damage and the role of the activated ATM/p53 signaling pathway in testicular toxicity. The present study showed that MC-LR exposure significantly reduced gonadal index and induced pathological damage of the testes in mice. In addition, MC-LR increased the oxidative stress-related indicator hydroxyl radical, accompanied by increased levels of DNA damage-related indicators gamma-H2AX, 8-hydroxy-2'-deoxyguanosine, the olive tail moment (OTM) and DNA content of comet tail (TailDNA%) in trailing cells. Moreover, MC-LR activated the ATM/p53 pathway by enhancing the phosphorylation levels of ATM, CHK2 and p53 proteins, and then led to cell autophagy, ultimately triggering disrupted testicular cell arrangement, reduced sperm count and spermatogenic cell shedding. Importantly, after pretreatment with the antioxidant NAC, the expression levels of DNA damage-related indicators and the extent of damage in male germ cells were significantly reduced. Furthermore, pretreatment with the ATM inhibitor KU55933 could reduce the occurrence of autophagy and mitigate testicular toxicity of MC-LR through inhibiting the activation of the ATM/p53 pathway. These results indicate that MC-LR-induced oxidative stress can activate the DNA damage-mediated ATM/p53 signalling pathway to induce autophagy in male germ cells. This study provides a novel insight to further clarify the reproductive toxicity caused by MC-LR and to protect male reproductive health.

A new identity of microcystins: Environmental endocrine disruptors? An evidence-based review.

Microcystins (MCs) are widely distributed cyanobacterial toxins in eutrophic waters. At present, the endocrine-disrupting effects of MCs have been extensively studied, but whether MCs can be classified as environmental endocrine disruptors (EDCs) is still unclear. This review is aimed to evaluate the rationality for MCs as to be classified as EDCs based on the available evidence. It has been identified that MCs meet eight of ten key characteristics of chemicals that can be classified as EDCs. MCs interfere with the six processes, including synthesis, release, circulation, metabolism, binding and action of natural hormones in the body. Also, they are fit two other characteristics of EDC: altering the fate of producing/responding cells and epigenetic modification. Further evidence indicates that the endocrine-disrupting effect of MCs may be an important cause of adverse health outcomes such as metabolic disorders, reproductive disorders and effects on the growth and development of offspring. Generally, MCs have endocrine-disrupting properties, suggesting that it is reasonable for them to be considered EDCs. This is of great importance in understanding and evaluating the harm done by MCs on humans.

Advances in the toxicology research of microcystins based on Omics approaches.

Microcystins (MCs) are the most widely distributed cyanotoxins, which can be ingested by animals and human body in multiple ways, resulting in a threat to human health and the biodiversity of wildlife. Therefore, the study on toxic effects and mechanisms of MCs is one of the focuses of attention. Recently, the Omics techniques, i.e. genomics, transcriptomics, proteomics and metabolomics, have significantly contributed to the comprehensive understanding and revealing of the molecular mechanisms about the toxicity of MCs. This paper mainly reviews current literature using the Omics approaches to explore the toxicity mechanism of MCs in liver, gonad, spleen, brain, intestine and lung of multiple species. It was found that MCs can exert strong toxic effects on various metabolic activities and cell signal transduction in cell cycle, apoptosis, destruction of cell cytoskeleton and redox disorder, at protein, transcription and metabolism level. Meanwhile, it was also revealed that the alteration of non-coding RNAs (miRNA, circRNA and lncRNA, etc.) and gut microbiota plays an essential regulatory role in the toxic effects of MCs, especially in hepatotoxicity and reproductive toxicity. In addition, we summarized current research gaps and pointed out the future directions for research. The detailed information in this paper shows that the application and development of Omics techniques have significantly promoted the research on MCs toxicity, and it is also a valuable resource for exploring the toxic mechanism of MCs.

IRE1 and CaMKKß pathways to reveal the mechanism involved in microcystin-LR-induced autophagy in mouse ovarian cells.

Microcystin-LR (MC-LR) is an emerging water pollutant produced by blooming cyanobacteria. It could be absorbed into human body via contaminated food and drinking water causing severe reproductive toxicity. Previous studies showed that MC-LR could regulate autophagy by inducing endoplasmic reticulum (ER) stress thereby causing female reproductive toxicity. However, the molecular mechanisms of MC-LR-induced autophagy remain to be elucidated. It is known that IRE1 and CaMKKß pathways are two important pathways involved in autophagy induced by ER stress. Hence, this study investigated the roles of both pathways in MC-LR-induced autophagy in mouse ovarian cells. The results showed that MC-LR significantly up-regulated the expression of autophagy marker proteins LC3Ⅱ and BECLIN1 and down-regulated the expression of P62 in vivo and in vitro. MC-LR-caused increase of autophagosomes could be observed in KK-1 cells by MDC staining. MC-LR induced the formation of autolysosomes as indicated by the overlap of LAMP1 and LC3. Meanwhile, MC-LR significantly activated the proteins in IRE1 pathway (IRE1, XBP1 and JNK) and in CaMKKß pathway (CaMKKß, AMPK, mTOR). Furthermore, MC-LR caused weight loss and ovarian histopathological damage in mice. In contrast, after the expression and function of IRE1 and CaMKKß were inhibited with siRNA in vitro and by inhibitors (4µ8C and STO-609, respectively) in vivo, the up-regulation of LC3Ⅱ and BECLIN1 and the degradation of P62 induced by MC-LR were significantly suppressed. MC-LR-induced autophagosomes in KK-1 cells and autolysosomes in mouse ovarian cells were also decreased. Moreover, the knockdown of IRE1 and CaMKKß relieved MC-LR-induced histopathological injury to mouse ovaries. These results indicated that MC-LR induced ovarian cell autophagy and ovarian injury via IRE1 and CaMKKß pathways. This study is the first study revealing the molecular mechanisms of MC-LR-induced autophagy of ovarian cells and providing new insights into the female reproductive toxicity of MC-LR.

Microcystin-leucine arginine exposure contributes to apoptosis and follicular atresia in mice ovaries by endoplasmic reticulum stress-upregulated Ddit3.

Microcystin-leucine arginine (MC-LR), an intracellular toxin to cause reproduction toxicity, is produced by blooming cyanobacteria and widely distributed in eutrophic waters. It is revealed that MC-LR-induced female reproductive toxicity is more severe than male reproductive toxicity. Previous studies mainly focused on male reproductive toxicity, and the molecular mechanisms of MC-LR-induced apoptosis, follicular atresia and infertility in female remain largely unclear. Here, it was found that MC-LR treatment could induce apoptosis, inflammation, follicular atresia, and decrease of gonadal index in mice ovaries. RNA-Seq data showed that the up-regulation of DNA-damage inducible transcript 3 (Ddit3) under endoplasmic reticulum (ER) stress had predominantly regulatory role in MC-LR-induced apoptotic pathway. Furthermore, MC-LR exposure promoted cleavage of activating transcription factor 6 (ATF6, 50kd), inositol-requiring enzyme 1 (Ire1) expression, phosphorylation of IRE1, mitogen-activated protein kinase 5 (Map3k5) and Ddit3 expression, which was accompanied by the upregulation of death receptor 5 (Dr5) and active-caspase-3, and a decrease in Bcl-2 expression. ER stress inhibitor 4-Phenyl butyric acid (4-PBA) ameliorated these MC-LR-induced changes in protein or mRNA level. More importantly, knockdown of Ddit3 suppressed MC-LR-induced cell apoptosis and follicular atresia by directly regulating Dr5 and Bcl-2. Additionally, it was also found that MC-LR increased Map3k5 phosphorylation by inhibiting protein phosphatase 2A (PP2A) activity, and then promoted Ddit3 expression. In short, our data suggests that Ddit3 promotes MC-LR-induced mice ovarian cells apoptosis and follicular atresia via ER stress activation, which provides a new insight into the relation between infertility in females and the emerging water pollutant MC-LR.