Differences in Abnormal Water Metabolism between SD Rats and KM Mice Intoxicated by Microcystin-RR.

The effects of microcystin-RR (MC-RR) on water metabolism were studied on Sprague-Dawley (SD) rats and KunMing (KM) mice. In the single dose toxicity test, polydipsia, polyuria, hematuria and proteinuria were found in group of rats receiving a MC-RR dose of 574.7 µg/kg, and could be relieved by dexamethasone (DXM). Gradient damage was observed in kidney and liver in rats with gradient MC-RR doses of 574.7, 287.3, and 143.7 µg/kg. No significant water metabolic changes or kidney injuries were observed in mice treated with MC-RR doses of 210.0, 105.0, and 52.5 µg/kg. In the continuous exposure test, in which mice were administrated with 140.0, 70.0, and 35.0 µg/kg MC-RR for 28 days, mice in the 140.0 µg/kg group presented increasing polydipsia, polyuria, and liver damage. However, no anatomic or histological changes, including related serological and urinary indices, were found in the kidney. In summary, abnormal water metabolism can be induced by MC-RR in rats through kidney injury in single dose exposure; the kidney of SD rats is more sensitive to MC-RR than that of KM mouse; and polydipsia and polyuria in mice exposed to MC-RR for 28 days occurred but could not be attributed to kidney damage.

Water metabolism dysfunction via renin-angiotensin system activation caused by liver damage in mice treated with microcystin-RR.

Microcystins (MCs) are a group of monocyclic heptapeptide toxins that have been shown to act as potent hepatotoxins. However, the observed symptoms of water metabolism disruption induced by microcystin-RR (MC-RR) or MCs have rarely been reported, and a relatively clear mechanism has not been identified. In the present study, male mice were divided into 4 groups (A: 140µg/kg, B: 70µg/kg,C: 35µg/kg, and D: 0µg/kg) and administered MC-RR daily for a month. On day 8 of treatment, an increase in water intake and urine output was observed in the high-dose group compared with the control, and the symptoms worsened with the repeated administration of the toxin until day 30. In addition, the urine specific gravity decreased and serum enzymes that can reflect hepatic damage increased in the high-dose group compared with the control (P<0.05). The mRNA level of angiotensinogen (AGT) in hepatocytes was upregulated to approximately 150% of the control (P<0.05), and the serum renin-angiotensin system (RAS) was activated in the high-dose group; however, signs of renal injury were not observed throughout the experiment. After the toxin treatment was completed, the high levels of the RAS and vasopressin in group A returned to normal levels within 1 week. As expected, the symptoms of polyuria and polydipsia also disappeared. Therefore, we propose that water metabolism dysfunction occurs via RAS activation caused by liver damage because the increased serum RAS levels in the experiment were consistent with the increased urine output and water intake in the mice during the observation period. In addition, we found for the first time that a RAS blocker could alleviate the observed polyuria and polydipsia and inactivate the high level of the RAS induced by MC-RR in a dose-dependent manner, which further supported our hypothesis.

[Isolation and cultivation of a wild microcytin-RR-producing cyanobacterium and verification of its toxin by high performance liquid chromatography and acute oral toxicity].

OBJECTIVE: Attempting to isolate and cultivate the microcytin-RR-producing cyanobacteria from natural blooms as well as to further investigate some characteristics of their growth and metabolite toxicity. METHODS: Capillary-pipette method was used to isolate wild Microcystis strains collected from eutrophicated lakes. The isolated strains were cultured in BG11 media at (25 ± 1) °C, under 2 000 lx illumination of fluorescent light with a light-dark rhythm of 12-12 h. The growth curve was observed by measuring optical density of culture suspension, toxin-related genes and the metabolite toxins were identified separately by PCR and HPLC, and its acute toxicity was carried out by orally administered toxins to Kunming (KM) mice. RESULTS: One of five toxigenic strains from 198 collected samples was confirmed to be a MC-RR producing blue-green alga by existing two specific toxin-synthesized enzyme genes and showing specific chromatographic peak of the toxin compared with standard MC-RR through both PCR and HPLC methods. The toxic strain was classified as Microcystin aeruginosa by morphologic and phylogenetic tree analysis. The growth length of the strain lasted nearly 81 days with 55-60 days' exponential phase and the maximal concentration of 5.52 × 107 cell/ml. The LD50 of the MC-RR to the KM mice ranged from 10.75 mg/kg to 13.45 mg/kg of body weight. As a result of the acute toxicity, the enzymatic indexes in serum such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) were significantly higher than those in the control group. The levels of ALT, AST, ALP and LDH in the treated group at 45 min were (157.08 ± 20.38), (333.00 ± 68.53), (392.70 ± 89.59) and (1 071.13 ± 160.22) U/L respectively, and at 4 h were (514.68 ± 156.87), (593.15 ± 40.41), (618.55 ± 208.76) and (2 281.72 ± 866.67) U/L respectively, and meanwhile the values of ALT, AST, ALP and LDH in the control group were (40.30 ± 4.89), (142.70 ± 26.59), (56.90 ± 11.89) and (509.50 ± 94.75) U/L separately (t values at 45 min were -11.20, -5.77, -7.38, -6.60 respectively, and at 4 h were -6.04, -20.21, -5.35, -4.07 respectively, P values were all <0.01). The liver coefficient in the treated group at 45 min and 4 h were 6.855 ± 0.225 and 8.409 ± 0.276, significantly higher than that (5.784 ± 0.286) in the control group (t values were -3.96 and -12.22, P values were both <0.01). The histopathological changes of liver were hyperemia obviously. CONCLUSION: Isolated from the bloom waters, a strain of Microcystis aeruginosa is obtained with characteristics of longer growth duration, positive microcystin synthetase genes, and dominant production of MC-RR. The LD50 of the extracted MC-RR administered by oral route to mice is (12.10 ± 1.35) mg/kg of body weight, and liver is the target organ of MC-RR. The existence and potential risk of MC-RR in China cannot be ignored.

[The influence of microcystin-LR on monocytes and lymphocytes of mice].

OBJECTIVE: To investigate the influence of microcystin-LR (MC-LR) on monocytes and lymphocytes in blood of mice and to find a sensitive index of toxic effects. METHODS: Specific pathogen free Kunming male mice, aging 1 month-old,were randomly divided into 5 groups by weights, 7 mice for each group. The mice in 5 groups were exposed to MC-LR through intraperitoneal injection at 0, 3.125,6.250, 12.500 and 25.000 µg/kg respectively for 7 days. Then cytokine levels in the serum were measured by radioimmunoassay, DNA-protein crosslinks (DPC) was measured by the SDS/KCl precipitation technique, and the phagocytosis and ROS of leukocytes were detected by flow cytometry. RESULTS: The levels of interleukin 6 in the 6.250, 12.500 and 25.000 µg·kg(-1)·d(-1) dose groups were (346.837 ± 25.536), (360.847 ± 37.886) and (434.245 ± 35.858)pg/ml respectively, which were significantly higher than those in the control group which the value was (232.775 ± 32.816) pg/ml (t values were -7.258, -6.760 and -10.966 respectively, P values were all < 0.05).While the level of tumor necrosis factor-alpha was(10.782 ± 0.966) fmol/ml in 25 µg·kg(-1)·d(-1) dose group was statistically lower than it in the control group which the value was (16.878 ± 3.378) fmol/ml (t value was 4.591, P < 0.05). The DPC levels of lymphocytes in 6.250, 12.500 µg·kg(-1)·d(-1) dose group were (242.576 ± 7.545),(241.472 ± 2.793) ng/ml,higher than it in the control group while the value was (228.657 ± 4.130) ng/ml (t value was -4.282, -6.801, P values were all <0.05). The fluorescence intensity of DCF in lymphocytes in the 4 treated groups were separately 3299.37 ± 120.54, 3281.38 ± 58.34, 3308.06 ± 136.12 and 3346.92 ± 108.69, all significantly lower than 3770.81 ± 131.39 in the control group (t values were 6.995, 9.007, 6.472 and 6.577 respectively, and P values were all <0.05). The fluorescence intensity of DCF in monocytes in the 4 treated groups (3271.51 ± 140.79, 3270.05 ± 117.92, 3326.90 ± 114.39 and 3292.49 ± 145.97 respectively) were also significantly lower than the value in the control group was 3841.72 ± 130.92 (t values were 7.847, 8.584, 7.835 and 7.411 respectively, P values were all <0.05). There was no significant difference in other index among the four experiment groups and the control group. CONCLUSION: The MC-LR administered via intraperitoneal injection to mice induced the alterations of some cytokines of monocytes and lymphocytes in blood. By comparison, the ROS of leukocyte was the most sensitive index.

[Purification and identification of microcystin-RR].

An effective method based on the combination of solid phase extraction and gel chromatography to extract and purify microcystin-RR (MC-RR) from natural cyanobacteria bloom was developed. Thirty five gram natural cyanobacterial bloom scum was extracted with 70% aqueous methanol. After the methanol in the crude extract was removed by a rotary evaporator, the solid phase extraction was used for preliminary purification and concentration. And then 7.5 mL eluent was concentrated to 2 mL and loaded onto a Sephadex LH-20 gel chromatographic column for further purification. The eluate was collected by tubes and detected at 238 nm. The elution curve was plotted by tube numbers as X-axis and the absorbance at 238 nm as Y-axis. The purity and the spectral characteristics of the final extract were identified with high performance liquid chromatograph (HPLC) and spectrophotometer, respectively. A total of 3.65 mg of MC-RR with a final yield of 74. 1% was obtained, and the purity was more than 90% with characteristic UV absorption spectra at 238 nm.

Comparison of response indices to toxic microcystin-LR in blood of mice.

In order to investigate the response indices to toxic microcystin-LR (MC-LR) in blood of mice, concentrations of free and total MC-LR in blood and tissues, accompanied by serous parameters in series including some enzymatic activities, hematology and the function of leukocytes, were determined in mice exposed to the toxin ranging from 3.125 to 25.000 µg kg(-1)day(-1) by intraperitoneal injection for 7 days. On the 7th day, the ratios of mass of free MC-LR in serum to the mass of MC-LR in given dose were 3.843-4.555%, while the ratios of total MC-LR in liver were 34.465-38.567%. Comparing the overall experimental results, the three most sensitive indices are total MC-LR in the liver, the phagocytic index and reactive oxygen species (ROS) which have shown significant differences between the lowest dose group and the control group. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and lactate dehydrogenase had proportional correlations with the MC-LR exposure doses, and the hematology of the majority of blood cells and the volume of erythrocytes were also influenced by the toxin. The alterations of some cytokines and the ROS of leukocytes were observed. The results of the studies suggest that measurement of MC-LR in blood is powerful and clear evidence to indicate that subjects have been exposed to MC-LR and can be used to discriminate from other causes leading to hepatic lesions although it is not as sensitive as other indices that are usually as useful complements to reflect the liver function.

[The establishment and application of the method with virus concentration and detection in drinking water].

OBJECTIVE: This study aimed to construct an effective method to concentrate and detect virus in drinking water, and human adenovirus pollution status in actual water samples was monitored by constructed method. METHODS: The concentration efficient of NanoCeram filter for the first concentration with source water and drinking water and the concentration efficient of the different concentrations of PEG 8000 for the second concentration were assessed by spiking f2 bacteriophage into water samples. The standard of human adenovirus for real-time PCR was constructed by T-A clone. The plasmid obtained was identified through sequence analyzing and consistency check comparing to target gene fragment was conducted by using blast algorithm. Then, real-time PCR was constructed to quantify the concentration of human adenovirus using the plasmid as standard. Water samples were concentrated by using NanoCeram filter on the spot and then concentrated for the second time by PEG/NaCl in 2011. The DNA of concentrated samples were extracted for the quantification of human adenovirus in real-time PCR subsequently to monitor the pollution of human adenovirus in water. RESULTS: For the first concentration by NanoCeram filter, the recovery rates were (51.63 ± 26.60)% in source water and (50.27 ± 14.35)% in treated water, respectively. For the second concentration, the highest recovery rate was reached to (90.09 ± 10.50)% at the concentration of 0.13 kg/L of PEG 8000. The sequence identity score of standard of adenovirus for real time PCR and adenovirus gene was 99%, implying that it can be successfully used to quantification with human adenovirus. The levels of human adenovirus in the water samples sampled in 2011 ranged from 4.13×10³ to 2.20×106 copies/L in source water, while range from 5.57×10² to 7.52×105 copies/L in treated water and the removal efficiency range was (75.49 ± 11.71)%. CONCLUSION: NanoCeram filers combined with PEG/NaCl was an effective method to concentrate virus in aquatic environment. There was a large number of human adenovirus in source water, and it is not sufficient to remove them thoroughly through conventional water treatment processes.

delta-Aminolevulinic acid dehydratase activity, urinary delta-aminolevulinic acid concentration and zinc protoporphyrin level among people with low level of lead exposure.

To evaluate the relationship of delta-aminolevulinic acid dehydratase (ALAD) activity, urinary delta-aminolevulinic acid (ALAU) level and blood zinc protoporphyrin (ZPP) concentration to low blood lead (PbB) levels, these biomarkers were determined for all subjects enrolled from a rural area of southeast China where people had low levels of exposure to lead. The mean values of PbB, ALAD, ALAU and ZPP were 67.11 microg/L (SD: 1.654, range: 10.90-514.04), 339.66 nmol ml(-1)h(-1) (1.419, 78.33-793.13), 20.64 microg/L (1.603, 2.00-326.00), and 0.14 micromol/L (3.437, 0.01-2.26), respectively. ALAD was inversely associated with low levels of PbB. ZPP was inversely related to low levels of PbB but positively related to relatively higher levels of PbB. Alcohol drinking contributed to low ALAD in men. Women had higher ZPP than men. ALAU had no significant association with PbB. In conclusion, ALAD possibly has a non-linear relation with low to moderate levels of PbB. At moderate levels of PbB, ZPP increases with increasing levels of PbB. ALAU is not suitable as an indicator for low levels of lead exposure.

[In vitro selection of specific aptamers against microcystin-LR].

OBJECTIVE: In vitro selection of specific RNA aptamers against microcystin-LR from a random RNA pool. METHODS: A RNA library with 40 randomized nucleotide positions was applied to select for specific aptamers to microcystin-LR covalently linked to Sepharose by using a standard in vitro selection protocol. RESULTS: The specific enriched RNA aptamer for microcystin-LR increased step by step from initial round to 11th round after which a plateau of the aptamer quantity was observed between 11th and 13th round. The enriched RNAs from last round were reverse transcribed, PCR amplified and cloned into E. coli DH10 b competent cells. Sixty colonies were sequenced from which 38 sequences were aligned and classified into 3 families and 5 duplicates and no conserved sequences were found among them. Eight representative clones from the groups were selected for further binding experiments comparing with original pool RNA. Four clone RNAs were identified with relatively high affinity to microcystin-LR, of which MC25 clone RNA could combine with microcystin-LR as lower as 0.5 micromol/L. CONCLUSION: Subpopulations of RNA molecules that bind specifically to microcystin-LR have been isolated from a population of random sequence RNA molecules, which might provide a new way for future application in environmental monitoring of microcystin.