Humans ; Male ; Semen ; Sperm Motility ; Mediation Analysis ; East Asian People ; Microcystins/toxicity* ; Spermatozoa ; Sperm Count ; Infertility, Male

Microcystin-leucine arginine (MC-LR), a potentially carcinogenic toxin, is produced by Cyanobacteria such as Microcystis and Ananabacteria during water bloom. Increasing evidence demonstrated that MC-LR induces male reproductive toxicity, mainly by inducing germ cell apoptosis, destroying cell cytoskeleton, interfering with DNA damage repair pathway, and damaging blood-testicular barrier (BTB), which eventually lead to male sterility. Testicular Sertoli cells are the somatic cells that directly contact with spermatogenic cells in seminiferous tubules. They not only regulate immune response to maintain testicular immune homeostasis by secreting a variety of cytokines and immunosuppressive factors, but also provide the protective effects of spermatogenic cells by forming BTB. MC-LR induces inflammation and apoptosis of Sertoli cells, and destroys the integrity of the BTB, and then causes spermatogenesis dysfunction.
Male ; Humans ; Sertoli Cells ; Leucine/pharmacology* ; Arginine/pharmacology* ; Microcystins/metabolism* ; Immunity

OBJECTIVEAttempting 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.

METHODSCapillary-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.

RESULTSOne 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.

CONCLUSIONIsolated 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.

Animals ; China ; Chromatography, High Pressure Liquid ; Cyanobacteria ; Hyperemia ; Lakes ; Liver ; Mice ; Microcystins ; Microcystis ; Phylogeny

OBJECTIVETo 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.

METHODSSpecific 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Animals ; Cytokines ; metabolism ; Lymphocytes ; drug effects ; Male ; Mice ; Mice, Inbred Strains ; Microcystins ; pharmacology ; Monocytes ; drug effects ; Reactive Oxygen Species ; metabolism

OBJECTIVETo explore the effects of microcystin-LR (MCLR) on the expression of base excision repair genes and genes related to apoptosis.

METHODSThe BRL-3A cells were exposed to different concentrations of MCLR for various periods of time and the cell viability was measured by MTT. The mRNA expression was determined with the quantitative real-time polymerase chain reaction (QRT-PCR).

RESULTSThe viability of BRL-3A cells significantly reduced in a concentration- and time-dependent manner. In 30 µg/ml group, the mRNA expression level (1.327 ± 0.028) of p53 increased significantly at 24 h after exposure, as compared with the other groups (1.005 ± 0.117, 0.862 ± 0.154, 1.028 ± 0.056 and 1.015 ± 0.091) (P < 0.05). The mRNA expression levels (5.080 ± 0.729, 5.820 ± 0.373, 6.018 ± 0.359 and 6.183 ± 0.515) of Bax in all exposure groups were significantly higher than that (1.024 ± 0.277) in control group at 24 h after exposure. However, the Bax mRNA expression level (0.604 ± 0.146) in the 30 µg/ml group at 72 h after exposure was significantly lower than those (1.004 ± 0.107, 0.811 ± 0.142, 0.855 ± 0.101 and 0.814 ± 0.056) in other groups (P < 0.05). When compared with control group (1.006 ± 0.132) and 1 µg/ml group (1.034 ± 0.241), the mRNA expression level (0.488 ± 0.147) of PARP1 in 30 µg/ml group at 48 h after exposure decreased significantly (P < 0.05). Furthermore, the mRNA expression levels (0.594 ± 0.180, 0.491 ± 0.015 and 0.305 ± 0.091) of JWA, XRCC1 and PARP1 in 30 µg/ml group at 72 h after exposure decreased significantly, as compared with the other groups (P < 0.05).

CONCLUSIONThe induction of gene expression is a transient phenomenon that occurred at different times of exposure for different genes. Inhibition of MCLR on the base excision repair gene expression may play important role in the course of MCLR promoting liver tumor.

Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; genetics ; Base Sequence ; Cell Line ; DNA Repair ; Gene Expression ; Microcystins ; toxicity ; RNA, Messenger ; genetics ; Rats

Animals ; Gene Expression ; Genes, p53 ; Liver ; drug effects ; metabolism ; pathology ; Liver Neoplasms ; metabolism ; pathology ; Male ; Microcystins ; toxicity ; Mutation ; Rats ; Rats, Wistar

OBJECTIVETo evaluate the effects of antagonistic action of epigallocatechin-3-gallate (EGCG) on microcystin LR (MC-LR) induced oxidative damage on mice and the expression of cytochrome P450 2E1 (CYP2E1) which was one of phase Iota detoxification enzymes.

METHODSA total of 24 specific pathogen free (SPF) male BALB/c mice were randomly divided into four groups, including control group, MC-LR group, low concentration EGCG group, and high concentration EGCG group. Mice were sacrificed on the 15th day, body weight, and the relative organ weight, liver antioxidant enzyme level and lipid peroxidation product, liver histopathology and CYP2E1 gene and protein expression were detected and analyzed respectively.

RESULTS(1) EGCG could antagonise the liver injury which had been damaged by MC-LR. (2) The malonaldehyde (MDA) level ((2.87 +/- 0.03) nmol/mg prot) and superoxide dismutase (SOD) level ((168.18 +/- 2.86) U/mg prot) in MC-LR group were significantly different when compared with the two EGCG treatment groups (the MDA values of the low and high concentration EGCG group were (2.37 +/- 0.05) nmol/mg prot and (1.44 +/- 0.05) nmol/mg prot, F = 906.63, P < 0.01; the SOD values were (176.55 +/- 2.98) U/mg prot and (184.89 +/- 1.53) U/mg prot, F = 32.32, P < 0.01). (3) MC-LR up-regulated the mRNA and protein expression of CYP2E1 (the mRNA values of MC-LR group and control were 1.41 +/- 0.26, 0.86 +/- 0.13, t = -4.22, P = 0.003; the protein values of MC-LR group and control were 0.24 +/- 0.03, 0.12 +/- 0.02, t = -9.21, P < 0.05). EGCG down-regulated the mRNA (the values of the low and high concentration EGCG group were 1.09 +/- 0.08, 0.99 +/- 0.09, F = 9.03, P = 0.004) and protein expression (the values of the low and high concentration EGCG group were 0.21 +/- 0.03, 0.14 +/- 0.02, F = 24.76, P < 0.05) of CYP2E1 which activated by MC-LR.

CONCLUSIONThe up-regulation of CYP2E1 which induced by MC-LR was inhibited by EGCG intervention. EGCG might antagonize the oxidation damage of hepatocytes in a certain degree.

Animals ; Catechin ; analogs & derivatives ; pharmacology ; Cytochrome P-450 CYP2E1 ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Microcystins ; adverse effects ; Oxidative Stress ; drug effects

OBJECTIVETo investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins (MCs) for further understanding hepatic intoxication and antioxidation mechanism in fish.

METHODSPhytoplanktivorous silver carp was injected intraperitoneally (i.p.) with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 microg MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection.

RESULTSThe damage to liver structure and the activities of hepatic antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxide (GPX) were increased in a time-dependent manner.

CONCLUSIONIn terms of clinical and histological signs of intoxication and LD50 (i.p.) dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also, the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.

Animals ; Antioxidants ; metabolism ; Carps ; metabolism ; Catalase ; metabolism ; Glutathione Peroxidase ; metabolism ; Injections, Intraperitoneal ; Liver ; drug effects ; enzymology ; pathology ; Microcystins ; administration & dosage ; isolation & purification ; pharmacology ; Phytoplankton ; physiology ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Survival Analysis ; Time Factors

OBJECTIVETo deeply explore the effects of microcystins (MC-LR) on Bax and Bcl-2 during the course of MC-LR promoting liver tumor.

METHODSapplied to set up the animal model, and the effect of MC-LR promoting liver tumor was evaluated by the Albertgamma-GT methods. And then, the immunohistochemical technique, RT-PCR and image analysis were used to study the expression of the Bcl-2 and Bax during the course of promoting tumor.

RESULTS(1) MC-LR might enhance the positive reaction rate of GGT. The positive reaction rate of GGT in DEN + pure toxin group was 100%, it was significantly higher than the DEN control group 22.22% (P < 0.05). (2) The intension and areas of the protein expression of Bcl-2 in DEN + pure toxin group were 0.0977 and 0.0315, and in DEN control group were 0.0460 and 0.0205, respectively. The expression level of Bcl-2 protein in DEN + pure toxin group were significantly higher than in DEN control group (P < 0.05). Simultaneously, the protein expression of Bax was significantly decreased by MC-LR (P < 0.05). The intension and areas of the expression of Bax in DEN + pure toxin group were 0.0283 and 0.0073, and in DEN control group were 0.0655 and 0.0244 respectively. (3) The mRNA expression of Bcl-2 was significantly increased by MC-LR. The intension of Bcl-2 mRNA expression in DEN + pure toxin group was 2.244, being significantly higher than in the other groups (P < 0.05). However, the mRNA expression of Bax showed no significant difference between DEN + pure toxin and the other groups.

CONCLUSIONThe expression change of Bcl-2 and Bax should possibly play an important role in the course of MC-LR promoting liver tumor.

Animals ; Apoptosis ; Carcinogens ; toxicity ; Hepatocytes ; metabolism ; Liver Neoplasms ; chemically induced ; metabolism ; Male ; Microcystins ; toxicity ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; bcl-2-Associated X Protein ; biosynthesis ; bcl-Associated Death Protein ; biosynthesis

OBJECTIVETo evaluate the antagonism effects of green tea (GT) against microcystin LR (MC-LR) induced hepatotoxicity and nephrotoxicity in mice.

METHODSAll 40 male mice were randomly divided into four groups. Mice in group III and IV were pretreated with green tea for free drink at doses of 2 g/L and 12 g/L prior to MC-LR intoxication, for consecutively 18 days. The toxin treatment mice were administered continually intraperitoneal injections of MC-LR at a dose of 10 microg x kg(-1) x d(-1) bw from day 6th till sacrifice, continually 13 days. Mice were sacrificed and immediately subjected to necropsy, and the body weight, relative organ weight, serum biochemical parameters, antioxidant enzyme levels (SOD and GSH), lipid peroxidation products (MDA) and histopathology were systematically evaluated.

RESULTSMC-LR exposure led to increase the oxidative stress and organ injury was significantly observed through biochemical parameters and microscopic evaluation. However, high dose of GT pretreatment caused a significant elevation in serum GSH and SOD levels, and a decrease of serum MDA level as compared with MC-LR control. The mean values of GSH and SOD activities were separately 467.29 mg/L and 139.22 U/ml in group IV. Subsequently, GT pretreatment obviously diminished the serum ALT, AST and Cr activities. Those pathological damages in liver and kidney, were to a certain extent, lessened in GT pretreatment mice in correlation with the biochemical parameters.

CONCLUSIONGT might elevate antioxidant defense system, clean up free radicals, lessen oxidative damages and protect liver and kidney against MC-LR induced toxicity.

Animals ; Antioxidants ; pharmacology ; Chemical and Drug Induced Liver Injury ; Free Radicals ; metabolism ; Kidney Diseases ; chemically induced ; metabolism ; pathology ; Liver Diseases ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred Strains ; Microcystins ; toxicity ; Oxidative Stress ; Tea