Cyanobacterial harmful bloom lipopolysaccharides: pro-inflammatory effects on epithelial and immune cells in vitro.

Cyanobacterial harmful blooms (CyanoHABs) pose a global ecological problem, and their lipopolysaccharides (LPS) are among the bioactive compounds they release. Previous studies on CyanoHAB-LPS from single cyanobacterial species have shown varying bioactivities in different in vitro cell models. In this study, we isolated LPS from 19 CyanoHAB samples collected at 18 water bodies in the Czech Republic over two consecutive seasons. The proportions of cyanobacteria, Gram-negative bacteria (G-), and other bacteria in the biomass were determined by qPCR, while the cyanobacterial genera were identified using light microscopy. In vitro models of keratinocytes (HaCaT), the intestinal epithelium (co-culture of differentiated Caco-2 cells and peripheral blood mononuclear cells - PBMC), and PBMC alone were treated with isolated LPS at concentrations of 50, 100, and 1 µg/ml, respectively. The endotoxin activities of these concentrations were within the range measured in the aquatic environment. Approximately 85-90% of the samples displayed biological activity. However, the potency of individual LPS effects and response patterns varied across the different in vitro models. Furthermore, the observed activities did not exhibit a clear correlation with the taxonomic composition of the phytoplankton community, the relative share of microbial groups in the biomass, endotoxin activity of the LPS, or LPS migration and staining pattern in SDS-PAGE. These findings suggest that the effects of CyanoHAB-LPS depend on the specific composition and abundance of various LPS structures within the complex environmental sample and their interactions with cellular receptors.

Retinoid-like compounds produced by phytoplankton affect embryonic development of Xenopus laevis.

Teratogenic effects, which were remarkably similar to those induced by retinoic acids, have been seen in wild frogs indicating possible source of retinoids in the environment. Recent studies indicate that some cyanobacterial species can contain teratogenic retinoic acids (RAs) and their analogues. Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis. The study investigated the effects of exudates (extracellular compounds) of two cyanobacteria species with retinoic-like activity and one algae species on embryonic development of amphibians. The retinoid-like activity determined by in vitro reporter gene assay reached 528ng retinoid equivalents (REQ)/L and 1000ng REQ/L in exudates of Cylindrospermopsis raciborskii and Microcystis aeruginosa, respectively, while algal exudates showed no detectable activity. Total mean of retinoid-like copounds into exudate was 35.6ng ATRA/mil.cells for M.aeruginosa and 6.71ng ATRA/mil.cells for C.raciborskii, respectively. Toxicity tests with amphibian embryos up to 96h of development were carried out according to the standard guide for the Frog Embryo Teratogenesis Assay Xenopus. Lowest observed effect concentrations (LOEC) of malformations (2.5-2.6µg/L REQ) were two times lower than LOEC for ATRA (5µg/L). The exudates of both cyanobacteria were indeed provoking diverse teratogenic effects (e.g. tail, gut and eyes deformation) and interference with growth in frogs embryos, while such effects were not observed for the algae. Xenopus embryos were also exposed to all-trans retinoic acid (ATRA) in concentration range (1-40µg/L) equivalent to the REQs detected in cyanobacterial exudates. ATRA (10µg/L) caused similar teratogenic phenotypes at corresponding REQs as cyanobacterial exudates. The study confirms the ability of some species of cyanobacteria to produce retinoids naturally and excrete them directly into the environment at concentrations which might have adverse influence on the development of amphibians.

Estrogenic activity and contributing compounds in stagnant water bodies with massive occurrence of phytoplankton.

Stagnant water bodies have generally received little attention regarding the presence of endocrine disruptive compounds, although they can integrate diverse pollutants from multiple different sources. Many compounds of anthropogenic as well as natural origin can contribute to the overall estrogenicity of surface waters and some of them can exhibit adverse effects on aquatic biota even in very low concentrations. This study focused on freshwater ponds and reservoirs affected by water blooms and determined the estrogenic activity of water by in vitro bioassay as well as concentrations of several important groups of estrogenic compounds (estrogenic hormones, alkylphenols, and phytoestrogens) by LC-MS/MS analyses. Estrogenic hormones were found at concentrations up to 7.1 ng.L-1, similarly to flavonoids, whose concentrations did not exceed 12.5 ng.L-1. Among alkylphenols, only bisphenol A and 4-tert-octylphenol were detected in levels reaching 100 ng.L-1 at maximum. Estrogenic activity of water samples varied from below the quantification limit to 1.95 ng.L-1. There does not seem to be any general causal link of the massive phytoplankton occurrence with the estrogenicity of water or concentration of phytoestrogens, since they showed no direct relationship with the phytoplankton abundance or composition across sites. The contribution of the analysed compounds to the estrogenic activity was calculated in three scenarios. In minimum scenario, just the compounds above quantification limit (LOQ) were taken into account and for most samples, only minor part (<6%) of the biological activity could be explained. In the mean and maximum scenarios, we included also compounds below LOQ into the calculations at the level of LOQ/2 and LOQ, respectively. In these cases, a considerable part of the estrogenic activity could be attributed to the possible presence of steroid estrogens below LOQ. However, for the samples with estrogenic activity greater than 1 ng.L-1, more than 50% of the estrogenic activity remained unexplained even in the maximum scenario. Probably other compounds or possible interactions between individual substances cause the estrogenic activity in these types of water bodies and in this case, the results of LC-MS/MS analyses cannot sufficiently predict the biological effects. A complex approach including bioassays is needed when assessing the estrogenicity of these types of surface waters.

Markers of acute stress in pigs.

This study explores the biological validation of markers of acute stress in pigs subjected to transportation for slaughter. The stress markers selected for monitoring were neopterin and cortisol. Their levels in pig serum were measured for two porcine stress syndrome genotypes, NN and Nn, after a 30-min transport to a slaughterhouse. Blood samples were withdrawn before transport (control group) and immediately after the animals' arrival (experimental group). The values of neopterin and cortisol measured before the transport were 5.60+/-1.65 nmol/l and 273.54+/-66.17 nmol/l respectively. After the transfer, the concentration of cortisol rose significantly compared to the control (355.69+/-85.13 nmol/l, p<0.01). Neopterin concentrations in the serum (8.25+/-1.60 nmol/l) were also significantly higher (p<0.01) after transportation. The elevated concentrations of both analytes were found to be independent of the genotype. These results document the stimulation of the endocrine system and the immune system that develops in animals undergoing transportation for slaughter.

Neopterin and biopterin as biomarkers of immune system activation associated with castration in piglets.

Recent reports have shown that stressful situations may affect the production of unconjugated pterins (neopterin and biopterin). The aim of the study was to investigate the effect of castration on neopterin and biopterin plasma concentrations in piglets, using 2 groups of 12 piglets allocated to castrated and uncastrated (control) groups. Pterin concentrations were determined by HPLC with fluorescence detection. Blood samples were also analzyed for leukocyte profiles and plasma cortisol concentrations. A time × treatment interaction (P < 0.05) was detected for neopterin concentrations, such that neopterin was greater (P < 0.01) at 1 h after surgery in castrated piglets compared with precastration concentrations, and neopterin was greater (P = 0.05) in castrated than in control piglets at 1 h. Castration had no effect on biopterin concentration (P > 0.1). Time effects (P < 0.05) for neutrophil and lymphocyte concentrations and neutrophil-to-lymphocyte ratios were found. A time × treatment interaction (P < 0.01) was detected for plasma cortisol concentrations, such that cortisol was greater (P < 0.01) at 1 and 24 h after surgery in castrated piglets compared with precastration concentrations and was greater (P < 0.01) in castrated than in control piglets at 1 and 24 h. This study showed that castration activated the immune system of piglets as demonstrated by an increase in plasma neopterin concentrations.