Ultrasonically induced degradation of microcystin-LR and -RR: identification of products, effect of pH, formation and destruction of peroxides

Microcystins (MCs) are a family of toxic peptides produced by a number of cyanobacteria commonly found in lakes, water reservoirs, and recreational facilities. The increased eutrophication of freshwater supplies has led to an increase in the incidence of cyanobacterial harmful algal blooms and concerns over the public health implications of these toxins in the water supply. Conventional water treatment methods are ineffective at removing low concentrations of cyanotoxins, hence specialized treatment is usually recommended for treatment of contaminated water. In this study, the products of ultrasonically induced degradation of microcystin-LR (MC-LR) and microcystin-RR (MC-RR) were analyzed by LC-MS to elucidate the probable pathways of degradation of these toxins. Results indicate preliminary products of sonolysis of MCs are due to the hydroxyl radical attack on the benzene ring and diene of the Adda peptide residue and cleavage of the Mdha-Ala peptide bond. The effect of pH on the toxin degradation was evaluated since the pH of the solution changes upon ultrasonic irradiation and varies with the water quality of treatable waters. The initial rate of MC-LR degradation is greater at acidic pH and coincides with the change in hydrophobic character of MC-LR as a function of pH. Hydrogen and organic peroxides are formed during ultrasonic irradiation, but can be eliminated by adding Fe(II). The addition of Fe(II) also accelerates the degradation of MC-LR, presumably by promoting the formation of hydroxyl radicals via conversion of ultrasonically produced H2O2. These findings suggest that sonolysis can effectively degrade MCs in drinking water.     

CE和HPLC测定水源水体中微囊藻毒素方法比较

利用毛细管电泳（capillary electrophoresis,CE）仪结合紫外-可见光二极管阵列检测器检测技术建立水体中痕量微囊藻毒素的检测新方法。通过与GB/T 20466-2006《水中微囊藻毒素的测定》高效液相色谱（high performance liquid chromatography,HPLC）方法对比分析,进行2 种检测技术的评价。CE检测条件为：毛细管柱（60 cm×75 μm i.d.）,有效长度为44 cm,分离缓冲溶液为12 mmol/L硼酸盐（pH 9.0）,分离电压25 kV,检测波长238 nm,压力6 895 Pa流体动力学进样;优化后的HPLC检测条件为：C18色谱柱（250 mm×4.6 mm i.d.,5 μm）,甲醇-磷酸盐缓冲溶液（60∶40,V/V）为流动相,检测波长238 nm,柱温度35 ℃,流速1 mL/min。结果表明：CE对3 种微囊藻毒素MC-RR、MC-LR和MC-YR的检出限分别是0.16、0.20 μg/mL和0.24 μg/mL,HPLC对3 种微囊藻毒素的检出限分别为0.020、0.079 μg/mL和0.052 μg/mL,这2 个方法的灵敏度相差1 个数量级;加标回收率分别92.5%～106.0%和99.6%～102.5%,CE对应的保留时间和峰面积的精密度相对标准偏差为0.53%～0.64%和2.67%～3.29%;HPLC法的保留时间和峰面积精密度相对标准偏差为0.16%～0.53%和0.80%～1.53%。检测同一水样中微囊藻毒素含量,CE检测结果和HPLC结果之间差异不显著（P＞0.05）。     

微囊藻毒素-LR对小鼠毒性机制的研究

目的：探讨微囊藻毒素-LR(MC-LR)引起小鼠机体损伤可能的作用机制。方法：通过腹腔注射单次给予小鼠30μg/kg bw 剂量的MC-LR,测定其在给药后1、4、8h 的脏器系数和一些血液生化指标。结果：短时间内(给药后4h)肝脏系数显著增加,肾脏系数变化不明显;血液CAT 含量显著上升,BUN 先显著上升再显著下降;CAT 活力显著上升;SOD 变化不明显。结论：MC-LR 首先攻击肝细胞,产生大量的自由基,破坏细胞膜,进而损伤了肝细胞,影响氨基酸的代谢,使肝脏充血、肿胀;接着又引发肾功能不全,使肾小管过滤能力降低;最后,随着时间的推移,对机体造成了潜在的永久伤害,但机体的修复功能使这种伤害表观上不可见,即MCLR对机体造成的损害在一定程度上是可逆的。     

Mechanistic study and the influence of oxygen on the photosensitized transformations of microcystins (cyanotoxins)

Microcystins (MCs) produced by cyanobacteria are strong hepatotoxins and classified as possible carcinogens. MCs pose a considerable threat to consumers of tainted drinking and surface waters, but the photochemical fate of dissolved MCs in the environment has received limited attention. MCs are released into the environment upon cell lysis along with photoactive pigments including phycocyanin and chlorophyll a. The concentrations of MCs and pigments are expected to be greatest during a bloom event. These blooms occur in sunlit surface water and thus MCs can undergo a variety of solar initiated or photosensitized transformations. We report herein the role of oxygen, sensitizer, and light on the photochemical fate of MCs. The phycocyanin photosensitized transformation of MCs is elucidated, and photosensitized isomerization plays an important role in the process. The UV-A portion of sunlight was simulated using 350 nm light and the phototransformations of three MC variants (-LR, -RR, -LF) were investigated. Singlet oxygen leads to photooxidation of phycocyanin, the predominant pigment of cyanobacteria, hence, reducing the phototransformation rate of MCs. The phototransformation rate of MC-LR increases as pH decreases. The pH effect may be the result of MCs association with phycocyanin. Our results indicate photosensitized processes may play a key role in the photochemical transformation of MCs in the natural water.     

Essential roles of p53 and MAPK cascades in microcystin-LR-induced germline apoptosis in Caenorhabditis elegans

Hepatotoxin microcystin-LR (MC-LR) can induce apoptosis in a variety of cells. However, the underlying pathways of MC-LR-induced apoptosis have not been well elucidated yet. To find out the roles of underlying pathways in apoptosis signaling in response to MC-LR, germ cell corpses were scored in Caenorhabditis elegans N2 wild type and strains carrying mutated alleles homologous to their mammalian counterparts. We found that exposure to MC-LR at 1.0 μg/L significantly increased germline apoptosis in N2. Germline apoptosis was absent at all doses in ced-3 and ced-4 loss-of-function strains. MC-LR-induced apoptosis was blocked in Bcl-2 gain-of-function strain ced-9(n1950), whereas it showed a slight increase in BH3-only protein EGL-1 mutated strain. The null mutation of cep-1, which is the homologue of p53 tumor suppressor gene, significantly inhibited MC-LR-induced cell death, and checkpoint proteins HUS-1 and CLK-2 exerted proapoptotic effects. Apoptosis in loss-of-function members of ERK, JNK, and p38 MAPK signaling pathways reduced significantly under MC-LR exposure, and members of MAPKK subgroup JKK-1, MEK-1, and SEK-1 worked cooperatively. Our results show that the caspase protein CED-3 and Apaf-1 protein CED-4 were absolutely required for the apoptotic processes, and that the p53/CEP-1 and MAPKs cascades played essential roles in modulating MC-LR-induced germline apoptosis in C. elegans.     

Yak milk–derived exosomal microRNAs regulate intestinal epithelial cells on proliferation in hypoxic environment

Intestinal epithelial cells (IEC) act as an important intestinal barrier whose function can be impaired upon induction by hypoxia. Although intestinal barrier injuries are preventable by milk-derived exosomal microRNAs (miRNAs), the underlying mechanism remains poorly understood. This study aimed to characterize the effect of yak and cow milk–derived exosomal miRNA on the barrier function of IEC-6 under hypoxic conditions, and explore the mechanism of yak milk exosomal miRNA to relieve the hypoxia stress. First, by Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) sequencing, the miRNA expression was systematically screened, and differential expression of 130 miRNAs was identified with 51 being upregulated and 79 downregulated in yak and cow milk–derived exosomes. Furthermore, the top 20 miRNAs that had a relatively consistent high expression in yak milk exosome were identified, and bta-miR-34a was found to be an effective regulator for alleviating hypoxic injury of IEC-6. In vitro assay of the role of bta-miR-34a on survival of IEC-6 in hypoxia by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) confirmed its effectiveness to significantly increase IEC-6 survival up to 13% for 12 h, and up to 9.5% for 24 h. Investigation on the regulatory relationship between bta-miRNA-34a and the hypoxia-inducible factor/apoptosis signaling pathway provided insights into the possible mechanisms by which bta-miR-34a activated the hypoxia-inducible factor and apoptosis signaling pathway, thus promoting IEC-6 survival. The results of this study suggest an important relationship between miRNA expression and intestine barrier integrity, which facilitated further understanding of the physiological function of yak and cow milk exosomal miRNAs, as well as mechanisms of hypoxia-driven epithelial homeostasis.     

Mechanisms and factors affecting sorption of microcystins onto natural sediments

The sorption of microcystins (MCs) to fifteen lake sediments and four clay minerals was studied as a function of sediment/clay properties, temperature, and pH through well-controlled batch sorption experiments. All sorption data for both sediments and clays are well described by a nonlinear Freundlich model (n(f) varies between 0.49 and 1.03). The sorption process for MCs exhibited different adsorptive mechanisms in different lake sediments mainly dependent on the sediment organic matter (OM). For sediments with lower OM (i.e., less than 8%), the sorption of MCs decreases with increasing OM and is dominated by the competition for adsorption sites between MCs and OM. In contrast, MC sorption to organic-rich (i.e., more than 8%) sediments increases with increasing OM and is dominated by the interaction between MCs and adsorbed OM. The sorption thermodynamics of MCs onto sediments showed that MC sorption is a spontaneous physisorption process with two different mechanisms. One mechanism is an exothermic process for sediment with lower OM, and the other is an endothermic process for sediment with higher OM. Furthermore, the sorption of MCs onto sediments is pH dependent (sorption decreased with increasing pH). These results provide valuable informations for a better understanding of the natural abiotic attenuation mechanisms for MCs in aquatic ecosystems.     

Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary

Little is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0-5.5 mm), pre-ovulatory follicles (6.0-7.0 mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular-luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular-luteal transition.     

Global gene expression profiling in larval zebrafish exposed to microcystin-LR and microcystis reveals endocrine disrupting effects of Cyanobacteria

Microcystis blooms occur worldwide and threaten aquatic ecosystems and human health. Sublethal effects on early developmental stages of fish are largely unknown, and research has mainly focused on microcystin toxins (such as MC-LR) rather than Microcystis cells. We exposed (96 h) zebrafish larvae to purified MC-LR (0-1000 μg/L) or lyophilized Microcystis aeruginosa containing 4.5 μg/L MC-LR and evaluated changes in global gene expression (Affymetrix GeneChip zebrafish genome arrays). Significant changes in gene expression (≥ 1.7-fold change, p < 0.0001) were determined with Rosetta Resolver 7.0, and ontology analysis was conducted with the DAVID bioinformatics tool. The number of differentially expressed genes relative to control increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR in mammals and older life stages of fish, as well as genes unique to larval zebrafish. Up-regulation of vitellogenin genes (vtg) (19.2-fold to >100-fold on arrays; 619.3-fold confirmed by quantitative PCR) was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR. Up-regulation of vtg indicates exposure to estrogenic substance(s) and suggests that Microcystis may be a natural source of environmental estrogens. Concerns about effects of Microcystis blooms may extend beyond those associated with the microcystin toxin.     

Drop coating deposition Raman (DCDR) for microcystin-LR identification and quantitation

A drop coating deposition Raman (DCDR) method was developed for the analysis of 2-200 ng samples of microcystin-LR (MC-LR), a ubiquitous and deadly hepatotoxin secreted by cyanobacteria. Solid phase extraction (SPE) of the toxin from a water sample enabled identification of MC-LR at 5 μg/L to 100 mg/L concentrations, and the collected results suggest lower detection limits can be readily attained following DCDR substrate modification. The DCDR process was applied to aqueous sample volumes of 0.5-20 μL that generated sample deposits from which MC-LR Raman spectra could be obtained within seconds. Larger volume samples were not required to improve spectral resolution. Volumes of 2 μL were ideal, producing "coffee-ring" MC-LR deposits that displayed distinct MC-LR Raman signals with high signal-to-noise within 1 s for a 200 ng sample and 300 s for a 2 ng sample. A linear correlation between Raman signal intensity and concentration was observed for 2-100 ng MC-LR samples after signal normalization. Reproducible MC-LR Raman spectra were collected from both fresh and aged samples. The presence of dissolved organic matter (DOM) did not preclude MC-LR identification in DCDR deposits of 3 μg of DOM mixed with 0.7 μg of MC-LR. Application of DCDR to environmental samples will require sample purification such as SPE before analysis, including critical cartridge wash and toxin rinsing steps. Raman based methods may one day facilitate simpler and faster sample throughput than traditional MC-LR detection methods.     

Monitoring algal toxins in lake water by liquid chromatography tandem mass spectrometry

Microcystins (MCs) and cylindrospermopsin (CYL) are potent natural toxins produced by cyanobacteria (blue-green algae) that grow worldwide in eutrophic freshwaters and cause animal and human water-based toxicoses. The main purpose of this work has been assessing the contamination levels of some MCs and CYL in eutrophic Italian lake (Albano) water. To do this, we have developed an original analytical method involving MC extraction with a sorbent (Carbograph 4) cartridge. CYL is a highly polar compound that is scarcely retained by any sorbent material. To analyze this toxin, we directly injected 0.5 mL of filtered lake water into the liquid chromatography (LC) column. Analytes were quantified by LC coupled to tandem mass spectrometry in the multireaction monitoring mode. The recovery of five selected MCs added to an analyte free lake water sample at three different concentrations (50, 150, and 500 ng/L) ranged between 93 and 103% with RSD values no larger than 8%. Limits of quantification (LOQ) of the five MCs were within the 2-9 ng/L range, whereas the LOQ of CYL was 300 ng/L. The occurrence and abundance of cyanotoxins in Lake Albano was monitored over four months (Sept-Dec 2004) by analyzing water samples collected monthly at the center of the lake and at different depths (from 0 to -30 m). During survey and with the MS/MS system operating in the parent ion scan mode, we individuated two demethylated forms of MC-RR and one demethylated variety of MC-LR. Demethylated MC-RRs are known to be even more toxic than MC-RR toward zooplanktic grazers. CYL was the most-abundant toxin during the first three monitoring months. To the best of our knowledge, this is the first work reporting concentration levels of CYL in lake water.     

Antibodies directed against a yeast carboxyl-terminal peroxisomal targeting signal specifically recognize peroxisomal proteins from various yeasts.

The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme (hydratase-dehydrogenase-epimerase) to peroxisomes of both Candida albicans and Saccharomyces cerevisiae (Aitchison,J.D., Murray, W.W. and Rachubinski, R. A. (1991).J. Biol. Chem. 266, 23197-23203). We investigated the possibility that this tripeptide may act as a general peroxisomal targeting signal (PTS) for other proteins in the yeasts C. tropicalis, C. albicans, Yarrowia lipolytica and S. cerevisiae, and in rat liver. Anti-AKI antibodies raised against the carboxyl-terminal 12 amino acids of trifunctional enzyme were used to search for this PTS in proteins of these yeasts and of rat liver. The anti-AKI antibodies reacted exclusively with multiple peroxisomal proteins from the yeasts C. tropicalis, C. albicans and Y. lipolytica. There was a weak reaction of the antibodies with one peroxisomal protein from S. cerevisiae and no reaction with peroxisomal proteins from rat liver. Antibodies directed against a synthetic peptide containing a carboxyl-terminal Ser-Lys-Leu PTS (Gould, S. J., Krisans, S., Keller, G.-A. and Subramani, S. (1990). J. Cell Biol. 110,27-34) reacted with multiple peroxisomal proteins of rat liver and with peroxisomal proteins of yeast distinct from those identified with anti-AKI antibodies. These results provide evidence that several peroxisomal proteins of different yeasts contain a PTS antigenically similar to that of C. tropicalis trifunctional enzyme and that this signal is absent from peroxisomal proteins from at least one mammalian system, rat liver.     

Effects of brown rice, rice bran, and polished rice on colon carcinogenesis in rats

Rice is a nutritious staple food with health-promoting activity. This study investigated the effects of brown rice, rice bran, and polished rice on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats. The colons were examined for preneoplastic lesions and the expression of pro-inflammatory proteins. Lipid peroxidation was determined in livers and plasma; antioxidant-associated parameters were determined in livers. The results showed that consumption of medium-level of rice bran significantly reduced the number of aberrant crypt foci (ACF) and altered their distribution. Brown rice and rice bran significantly reduced cyclooxygenase-2 (COX-2) expression of the middle colon. Brown rice, rice bran, and polished rice had no significant effect on plasma and hepatic lipid peroxides and hepatic antioxidant-associated parameters. Thus, rice bran may be one candidate of the active rice fraction that protects the colon against DMH-induced early carcinogenesis in rats and may be a novel dietary supplement for chemoprevention of colon cancer.     

A C/T mutation in microRNA target sites in BMP5 gene is potentially associated with fatness in pigs

Bone morphogenetic protein 5 (BMP5) gene was suggested to be a potential positional candidate gene for fatness trait QTLs on SSC7. Here by comparative sequencing of BMP5 gene in Meishan (MS) and Large White (LW) pigs, one SNP *131C>T in 3' un-translated region was detected. Association analysis results in 322 LW×MS F(2) pigs showed that CC pigs had thinner back fatness and heavier ham than TC (P<0.05 or P<0.1), and had a higher fat percentage and a lower lean meat percentage (P<0.1) than TT. Moreover, this C/T transition was predicted to alter BMP5 interaction with let-7c and miR-184 by using RNA22 and RNAhybrid. The negative expression of BMP5 gene with let-7c and miR-184 detected from miRNAs overexpression in swine fibroblast, indicating these 2 miRNAs might participate in the translational inhibition of BMP5 gene. Overall, SNP *131C>T might be a good marker for fatness traits.