Genotoxicity Induced By Microcystin-LR In Vitro And In Vivo

INTRODUCTION: The occurrence of toxic water blooms ofcyanobacteria (blue-green algae) is an increasing environmental hazard and causing a serious public health problem in many parts of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacterium microcystis aeruginosa which has potent hepatotoxicity and tumor promoting activity.OBJECTIVE: To investigate the genotoxicity of MCLR in vitro and invivo, and explore its potential toxicity mechanism.METHODS:( I ) We investigated the in vitro genotoxicity of MCLR in human lymphoblastoid cell line TK6. After short term (4h) as well as long term (24h) treatment with MCLR , cytotoxicity test, micronucleus assay and thymidine kinase (tk) gene mutation assay were conducted. Spontaneous and microcystin-LR- induced TK-deficient mutants were isolated, LOH analysis was carried out.(II) To assess the in vivo genotoxicity of MCLR, Trangenic mice (Muta?Mouse) were used to examine the mutant frequency in the lacZ and cII gene of liver and lung . The frequency of micronucleated reticulocytes (MNRETs) in peripheral blood were evaluated and mutation spectra of cII gene were analyzed. Co-mutagenic effect of MCLR by the combination with N-nitrosodiethylamine (DEN) in transgenic mutation assay was alsoinvestigated.(HI) Atlas?Glass Human 1.0 cDNA Microarray was used to examine effects of MCLR on 1090 genes expression of TK6 cells.RESULTS:( I) In contrast to negative response in 4h treatment, 24h treatment of TK6 cells with MCLR resulted in a linear dose response with mutant frequencies of up to 5-fold over control. The long-term treatment also induced micronuclei at 80 ug/ml. DNA isolated from mutants in the control culture and the 80|ag/ml MCLR treatment group was analyzed for loss of heterozygosity (LOH) using allele-specific PCR. The percentage of hemizygous LOH in MCLR-induced mutants (41.7 %) was two times that of the control (20.7%).(II) MCLR did not increase either MFs of the target genes in liver and lung or micronucleus frequency in the peripheral blood cells of the X/lacZ transgenic mouse. MFs were increased significantly in DEN treated mice, and the co-administration of MCLR did not potentiate its mutagencity. The sequence alterations of the c//gene in MCLR-treated group were not observed whereas the DEN treatment increased the incidence of A:T to T:A transversions (2-28%) and decreased G:C to A:T transitions (50-24%).(El) MCLR treatment up-regulated the expression of teratocarcinoma-derived growth factor 1 (TDGF1) or epidermal growth factor-like CRIPTO protein 1 (CR1) meanwhile down-regulated the expression of protein phosphatase receptors - KIAA0283 of TK6 cells.CONCLUSIONS:1. MCLR is a mutagen and clastogen in TK6 human lymphoblastoid cells after 24h treatment in vitro. It induced LOH, but not point mutations. The genotoxic activity may have been associated with the inductions of DSBs and /or its promoting activity.2. MCLR has no in vivo genotoxicity in X/lacZ transgenic mouse as it failed to induce gene mutation and micronucleus.3. Tumor promoting effects of MCLR is independent of its interaction to DNA as lack of potentiation of DEN- induced mutations in transgenes was observed.4. Down-regulating the expression of protein phosphatase receptor meanwhile up-regulating that of teratocacinoma-derived growthfactor l(TDGFl) may be potential tumor promotion mechanism of MCLR.