Cylindrospermopsin induces oocyte maturation and disrupts gene expression in zebrafish ovarian follicles.

There is evidence that cylindrospermopsin (CYN) exerts reproductive toxicity in mice. However, little information is available concerning the toxicity of CYN in nonmammalian vertebrates. Here, we investigated the direct action of CYN on female reproduction by studying germinal vesicle breakdown, transcript abundance, caspase-3 activity, and testosterone production using cultured follicle-enclosed zebrafish oocytes as a model system. Treatment of follicles with 1,000 µg/L CYN significantly increased GVBD, Caspase-3 activity, and hCG-induced testosterone secretion. Exposure to CYN also reduced the abundance of 3ßhsd as well as hCG-induced fshr and era transcripts and increased cyp19a1 mRNA levels. In summary, this study provides a framework for a better understanding of the adverse action of CYN on female reproduction in zebrafish and other vertebrate species. The findings are also relevant to developing valid biomarkers for CYN by measuring zebrafish oocyte maturation and gene expression.

Cylindrospermopsin Disrupts Estrous Cycle and Increases Spermatogenesis in Mice.

Cylindrospermopsin (CYN) is a water-soluble cyanotoxin that has been linked to several cases of poisoning in the world. In vitro studies have shown that CYN acts as an endocrine disruptor by inhibiting progesterone synthesis in primary cell cultures of women, showing estrogenic activity. However, in vivo assessment of CYN in the female and male reproductive systems remains unknown. We thus aimed to evaluate the in vivo effects of CYN in both the female and male reproductive systems of mice. A single intraperitoneal exposure to 64 µg of CYN/kg body weight was performed in females. Estrous cycle was evaluated daily by vaginal cytology, and serum progesterone and estradiol levels were measured after 50 days. We showed an impairment in the estrous cycle as well as a decrease in circulating plasma progesterone levels. In males, weekly intraperitoneal doses of 20 µg of CYN/kg body weight were given and groups were killed after one, two, or four doses. CYN increased the testosterone levels in the groups that received one or two doses of CYN. Additionally, CYN induced a transient increase in spermatozoa in males after four doses. Our results highlight that CYN interferes with both male and female reproductive systems and may lead to infertility. As far as we know, this is the first report showing the impacts of CYN on the mammalian reproductive system, suggesting a threat from this cyanotoxin to human and environmental health.

Degradation of Multiple Peptides by Microcystin-Degrader Paucibacter toxinivorans (2C20).

Since conventional drinking water treatments applied in different countries are inefficient at eliminating potentially toxic cyanobacterial peptides, a number of bacteria have been studied as an alternative to biological filters for the removal of microcystins (MCs). Here, we evaluated the degradation of not only MCs variants (-LR/DM-LR/-RR/-LF/-YR), but also non-MCs peptides (anabaenopeptins A/B, aerucyclamides A/D) by Paucibactertoxinivorans over 7 days. We also evaluated the degradation rate of MC-LR in a peptide mix, with all peptides tested, and in the presence of M. aeruginosa crude extract. Furthermore, biodegradation was assessed for non-cyanobacterial peptides with different chemical structures, such as cyclosporin A, (Glu1)-fibrinopeptide-B, leucine-enkephalin, and oxytocin. When cyanopeptides were individually added, P. toxinivorans degraded them (99%) over 7 days, except for MC-LR and -RR, which decreased by about 85 and 90%, respectively. The degradation rate of MC-LR decreased in the peptide mix compared to an individual compound, however, in the presence of the Microcystis extract, it was degraded considerably faster (3 days). It was noted that biodegradation rates decreased in the mix for all MCs while non-MCs peptides were immediately degraded. UPLC-QTOF-MS/MS allowed us to identify two linear biodegradation products for MC-LR and MC-YR, and one for MC-LF. Furthermore, P. toxinivorans demonstrated complete degradation of non-cyanobacterial peptides, with the exception of oxytocin, where around 50% remained after 7 days. Thus, although P. toxinivorans was previously identified as a MC-degrader, it also degrades a wide range of peptides under a range of conditions, which could be optimized as a potential biological tool for water treatment.

Comparative bioaccumulation and effects of purified and cellular extract of cylindrospermopsin to freshwater fish Hoplias malabaricus.

Many tropical freshwater ecosystems are impacted by cyanobacteria blooms increasing the risk of cyanotoxins exposure to aquatic organisms while human populations may be exposed by eating fish, drinking water, or dermal swimming. However, few toxicological data are available on the influence of cyanobacteria blooms in particular, cylindrospermopsin (CYN) on Brazilian neotropical fish. A number of studies demonstrated the ability of CYN to bioaccumulate in freshwater organisms and consequently enter the human food chain. The aim of the current study was to examine the effects of CYN following single intraperitoneal injection (50 µg/kg) of purified CYN (CYNp) or aqueous extract of CYN-producing cyanobacteria extract (CYNex) after 7 or 14 days. Biomarkers such as histopathology (liver), oxidative stress (liver and brain), and acetylcholinesterase (AChE) activity (muscle and brain) were utilized in order to assess the influence of CYN on Hoplias malabaricus. In terms of AChE activity, administration of CYNex and CYNp both muscle and brains were used as target tissues. In brain an increase of glutathione S-transferase (GST) activity and lipid peroxidation (LPO) levels was noted suggesting an imbalance in redox cycling. The majority of biomarkers did not present significant alterations in liver, but an elevation in superoxide dismutase (SOD) and glucose 6 phosphate dehydrogenase (G6PDH) activities was found. Different profiles of GST activity were observed in both studied groups (CYNex and CYNp) while LPO (CYNex and CYNp) and protein carbonylation (PCO) (CYNp) levels increased after exposure to CYN. The incidence of necrosis, melanomacrophages centers, and free melanomacrophages were detected as evidence of cell death and immune responses. Nonprotein thiols (NPT) levels were not markedly affected in both exposed groups. Data demonstrated that in vivo exposure to CYN produced biochemical and morphological disturbances in liver and brain of H. malabaricus.

Single and combined effects of microcystin- and saxitoxin-producing cyanobacteria on the fitness and antioxidant defenses of cladocerans.

Cyanobacteria produce different toxic compounds that affect animal life, among them hepatotoxins and neurotoxins. Because cyanobacteria are able to produce a variety of toxic compounds at the same time, organisms may be, generally, subjected to their combined action. In the present study, we demonstrate the single and combined effects on cladocerans of cyanobacteria that produce microcystins (hepatotoxins) and saxitoxins (neurotoxins). Animals were exposed (either singly or combined) to 2 strains of cyanobacteria isolated from the same environment (Funil Reservoir, Rio de Janeiro, Brazil). The effects on clearance rate, mobility, survivorship, fecundity, population increase rate (r), and the antioxidant enzymes glutathione-S-transferase (GST) and catalase (CAT) were measured. Cladoceran species showed a variety of responses to cyanobacterial exposures, going from no effect to impairment of swimming movement, lower survivorship, fecundity, and general fitness (r). Animals ingested cyanobacteria in all treatments, although at lower rates than good food (green algae). Antioxidant defense responses were in accordance with fitness responses, suggesting that oxidative stress may be related to such effects. The present study emphasizes the need for testing combined actions of different classes of toxins, because this is often, and most likely, the scenario in a more eutrophic world with global climatic changes. Environ Toxicol Chem 2017;36:2689-2697. © 2017 SETAC.

Low concentrations of cylindrospermopsin induce increases of reactive oxygen species levels, metabolism and proliferation in human hepatoma cells (HepG2).

Human hepatoma cells (HepG2) were exposed to purified cylindrospermopsin (CYN), a potent toxicant for eukaryotic cells produced by several cyanobacteria. CYN was not toxic at concentrations up to 10 µgl(-1), leading to increased viability and metabolism in cells cultured with 10% fetal bovine serum (FBS). Reduction of FBS concentration to 2% and induction of cytochrome P450 (CYP) isoforms were performed in order to make xenobiotic-metabolizing capacity of HepG2 cells closest to that of 'normal' cells. HepG2 cells proliferated less after CYPs-induction, and this induction has lead to similar results of non-induced cells, except for few individual parameters such lipid peroxidation. Foremost, low concentrations of CYN (below or equal 10 µgl(-1)) have induced HepG2 cells proliferation and metabolism increase, which was not expected.

A rapid bioassay for detecting saxitoxins using a Daphnia acute toxicity test.

Bioassays using Daphnia pulex and Moina micrura were designed to detect cyanobacterial neurotoxins in raw water samples. Phytoplankton and cyanotoxins from seston were analyzed during 15 months in a eutrophic reservoir. Effective time to immobilize 50% of the exposed individuals (ET50) was adopted as the endpoint. Paralysis of swimming movements was observed between approximately 0.5-3 h of exposure to lake water containing toxic cyanobacteria, followed by an almost complete recovery of the swimming activity within 24 h after being placed in control water. The same effects were observed in bioassays with a saxitoxin-producer strain of Cylindrospermopsis raciborskii isolated from the reservoir. Regression analysis showed significant relationships between ET50 vs. cell density, biomass and saxitoxins content, suggesting that the paralysis of Daphnia in lake water samples was caused by saxitoxins found in C. raciborskii. Daphnia bioassay was found to be a sensitive method for detecting fast-acting neurotoxins in natural samples, with important advantages over mouse bioassays.