Effects of Cyanobacterial Lipopolysaccharides from Microcystis on Glutathione-Based Detoxification Pathways in the Zebrafish (Danio rerio) Embryo

Abstract

Cyanobacteria (“blue-green algae”) are recognized producers of a diverse array of toxic secondary metabolites. Of these, the lipopolysaccharides (LPS), produced by all cyanobacteria, remain to be well investigated. In the current study, we specifically employed the zebrafish (Danio rerio) embryo to investigate the effects of LPS from geographically diverse strains of the widespread cyanobacterial genus, Microcystis, on several detoxifying enzymes/pathways, including glutathione-S-transferase (GST), glutathione peroxidase (GPx)/glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), and compared observed effects to those of heterotrophic bacterial (i.e., E. coli) LPS. In agreement with previous studies, cyanobacterial LPS significantly reduced GST in embryos exposed to LPS in all treatments. In contrast, GPx moderately increased in embryos exposed to LPS, with no effect on reciprocal GR activity. Interestingly, total glutathione levels were elevated in embryos exposed to Microcystis LPS, but the relative levels of reduced and oxidized glutathione (i.e., GSH/GSSG) were, likewise, elevated suggesting that oxidative stress is not involved in the observed effects as typical of heterotrophic bacterial LPS in mammalian systems. In further support of this, no effect was observed with respect to CAT or SOD activity. These findings demonstrate that Microcystis LPS affects glutathione-based detoxification pathways in the zebrafish embryo, and more generally, that this model is well suited for investigating the apparent toxicophore of cyanobacterial LPS, including possible differences in structure-activity relationships between heterotrophic and cyanobacterial LPS, and teleost fish versus mammalian systems.

Glutathione and zebrafish: Old assays to address a current issue

“…For example, exposure to antidepressant amitriptyline resulted in a U-shape response, such that lower concentrations decreased GSH concentration, while higher concentrations increased GSH concentration (Yang et al, 2014). Exposures to bacterial lipopolysaccharides (Jaja-Chimedza et al, 2012) and total particulate matter from cigarette smoke (Massarsky et al, 2015) also appeared to elevate GSH, while exposures to β-naphthoflavone and α-naphthoflavone (Timme-Laragy et al, 2009) did not significantly affect GSH concentrations.…”

High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance of Intact Zebrafish Embryos Detects Metabolic Changes Following Exposure to Teratogenic Polymethoxyalkenes from Algae

“…10,11,[13][14][15][16] Briefly, 100 embryos (12 h postfertilization [hpf]) per replicate (n = 4) were exposed in 35-mm-diameter polystyrene dishes to 20 lg/mL of PMA (in E3 medium 21 ) for 24 h. Exposure concentration was based on previously determined teratogenicity of PMA and specifically represent concentrations that are not acutely toxic (i.e., teratogenic or lethal) to minimize metabolic changes associated with mortality or severe development deformities.…”

“…The instrument is equipped with a 4-mm HR-MAS dual inverse 1 H/ 13 C probe with a magic-angle gradient. All measurements were carried out at a magic-angle spinning rate of 6 kHz and a temperature of 277 K. Temperature was controlled by a Bruker BVT3000 control unit.…”

“…With respect to imaging, MRI techniques utilize, in particular, the abundance of water in biological systems, coupled with the detection of the magnetic nuclear spin of hydrogen ( 1 H) atoms in H 2 O, to collect highresolution spatial data within biological samples and subsequently generate resolved, three-dimensional (3D) images. Conversely, NMR spectroscopy is capable of discerning particular molecules based, likewise, on the spin of 1 H or, in fact, isotopes of several relevant atoms (e.g., 13 C, 15 N), and more exactly, the corresponding chemical shift of a particular atom within the molecule. When applied to biological systems and the detection and measurement of metabolites in particular, NMR spectroscopy can be specifically enabled by a technique called high-resolution magic angle spinning (HR-MAS).…”

“…[7][8][9] In particular, embryonic and subsequent larval stages have proven particularly useful for characterizing teratogenicity (i.e., developmental toxicity). 8,9 With respect to the current study, the zebrafish embryo teratogenicity assay (ZETA) has been specifically used in the investigation of toxic, and specifically, teratogenic metabolites from marine and freshwater algae, including cyanobacteria, 10 and has enabled both characterization of known algal toxins [11][12][13] and identification (through screening, bioassay-guided isolation, and chemical/toxicological characterization) of otherwise unknown toxic metabolites. [14][15][16] In one such study, ZETA was used to identify a family of teratogenic secondary metabolites, namely, the polymethoxy-1-alkenes (PMAs), and subsequently demonstrate a taxonomically widespread distribution of these metabolites among both prokaryotic cyanobacteria and eukaryotic algae (i.e., Chlorophyta or green algae).…”