title: [Isolation and cultivation of a wild microcytin-RR-producing cyanobacterium and verification of its toxin by high performance liquid chromatography and acute oral toxicity].
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Environmental Medicine, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, MOE Key Lab of Environment and Health, Wuhan 430030, China.
authors.forename: Wenqing
authors.initials: W
authors.lastname: Xiao
authors.forename: Yonglu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.forename: Xianing
authors.initials: X
authors.lastname: Huang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhong
authors.forename: Weiguang
authors.initials: W
authors.lastname: Wang
authors.affiliation: Email: hygkd@mails.tjmu.edu.cn.
authors.forename: Kangding
authors.initials: K
authors.lastname: Gu
mesh_terms: D000818:Animals; D002681:China; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria; D006940:Hyperemia; D060106:Lakes; D008099:Liver; D051379:Mice; D052998:Microcystins; D046931:Microcystis; D010802:Phylogeny
chemical_list: D052998:Microcystins; C078588:microcystin