Protection against the toxicity of microcystin-LR and cylindrospermopsin in Artemia salina and Daphnia spp. by pre-treatment with cyanobacterial lipopolysaccharide (LPS)

Purified cyanobacterial lipopolysaccharide (LPS) was not acutely toxic to three aquatic invertebrates (Artemia salina, Daphnia magna and Daphnia galeata) in immersion trials. However, pre-exposure (24 h) to 2 ng mL⁻¹ LPS increased the LC₅₀ of microcystin-LR significantly in all 3 species. Similar results were observed with A. salina pre-treated with the same concentration of cyanobacterial LPS and subsequently exposed to cylindrospermopsin, increasing the LC₅₀ by 8. The findings indicate the need to include exposures to defined combinations of cyanotoxins, and in defined sequences, to understand the contributions of individual cyanotoxins in accounting for cyanobacterial toxicity to invertebrates in natural aquatic environments.     

Microcystin-LR affects cytoskeleton and morphology of rabbit primary whole embryo cultured cells in vitro

Microcystin-LR is the most frequently studied cyclic heptapeptide produced by different genera of cyanobacteria and is hepatotoxic to livestock and human populations. The adverse effects of microcystin-LR on morphology and cytoskeletal elements in different stages of early embryonal development have been studied in vitro. Embryos and whole embryo cultures have been exposed to microcystin-LR (10–100 μM). Actin filaments were visualized by fluorescence staining and the microtubular network labelled by immunostaining. Growth, development and cytoskeleton organization of the embryos embedded in zona pellucida are not affected by microcystin-LR in concentrations up to 100 μM, while whole embryo cell cultures are affected by the presence of microcystin-LR in the culture medium. High microcystin-LR concentrations (100 μM) cause cells to be detached and destroyed, while lower concentrations (10–20 μM) profoundly affect actin and microtubule organization. These effects are confirmed also by the presence of transformed microcystin-LR in all the media at the lowest concentrations. It seems that the changes to the cells are far more serious than that expressed in cell morphology. From our experiments we conclude that the presence of zona pellucida is an effective way of embryo protection against xenobiotics like microcystin-LR.     

The use of Lepidium sativum in a plant bioassay system for the detection of microcystin-LR.

Toxin-producing cyanobacteria pose a worldwide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. Detection of microcystins in water generally relies on specialised equipment and a delay of several days for transport and analysis. Little work has, however, been done on establishing a simple, cost-effective and sensitive plant bioassay for the detection of microcystin-LR (MCLR) in water at the WHO Tolerable Daily Intake guideline level of 1 microg/l. We investigated the effect of a MCLR extract at 1 and 10 microg/l on the growth of Lepidium sativum over 6 days. Exposure to 10 microg/l MCLR resulted in a significant decrease in root and leaf lengths and fresh weights of seedlings when compared to the controls. These results were consistent with seedlings exposed to pure MCLR at 10 microg/l. Seedlings exposed to 1 microg/l MCLR showed a significant decrease in root development from day 2 to day 6. Glutathione S-transferase and glutathione peroxidase activities were also significantly raised in plants from days 5 and 4, respectively, at both toxin levels investigated.     

Most cited papers in Toxicon.

Citation of a published work is one of the parameters considered in the analysis of relevance and importance of scientific contributions. In 2002, for the first time the Impact Factor of Toxicon has risen above 2.0, placing it at the 17th position among 76 journals in the 'toxicology' field. The aim of this article was to identify the most cited articles in Toxicon, that have contributed to the steady increase of its Impact Factor. The number of citations, complete reference and type of all documents appearing in Toxicon in the period 1963-2003 were retrieved from the ISI Web-of-Science homepage. The documents retrieved were sorted by the number of citations received. A 'citation index', defined as the number of citations divided by the number of years since publication, was calculated for each document. It was clearly seen that reviews in Toxicon received 4.4-fold more citations than articles. Unexpectedly, it was found that recent papers were proportionally more cited than old ones. A decrease in the proportion of papers dealing on 'snake*' through out the period and the broadened range of subjects of the most cited papers recently published in Toxicon reflects an increased 'visibility' in other fields of toxinology. Research on plant toxins gained its own space in Toxicon with newer publications showing high citation indexes. It can be postulated that these facts helped to increase Toxicon's Impact Factor from 1.248 in 1999 to 2.003 in 2002. With the increased number of issues in Toxicon as well as publications of subject-dedicated volumes containing mostly reviews, the Impact Factor of Toxicon is expected to keep rising in the near future.     

Freshwater Cyanobacterial Blooms and Primary Liver Cancer Epidemiological Studies in Serbia

A large part of Central Serbia experiences continual shortage of sufficient ground water resources. For that reason, more than 20 reservoirs serve as drinking water suppliers. Significant and persistent cyanobacterial “blooms” have been recognized in nine of them. Samples for cyanotoxin analyses were taken during and after “blooms” in ?elije Reservoir and from Kru?evac town-supplied tap water from that reservoir two days later. Concentration of microcystin-LR was 650 μ gL^–1 in the reservoir, while the tap water contained 2.5 μ gL^–1.

In the two investigated periods, the high primary liver cancer (PLC) mortality of 11.6 from 1980–1990 and extremely high PLC incidence of 34.7 from 2000–2002 were observed in the regions affected by heavy cyanobacterial “blooms.” In contrast, PLC mortality and incidence rates were substantially lower in the regions not affected by cyanobacterial blooms: in 1980–1990 the rate of PLC mortality amounted to 2.7 in Kosovo, 7.6 in Vojvodina, and 8.3 in the non-affected regions of Central Serbia; while in 2000–2002 PLC incidence amounted to 4.1 in Kosovo, 5.2 in Vojvodina, and 13.6 in the non- or less-affected regions of Central Serbia. Keeping in mind that the most affected PLC regions in Central Serbia (Topli?ki, Ni?ki, and ?umadijski regions) have the water supply systems based on six reservoirs found regularly in bloom during summer months and that some of the regions are also connected with two boundary “blooming” reservoirs, representing a total of eight of nine blooming reservoirs, it is easy to presume that the PLC incidence could be related to drinking water quality.

The uneven geographic distribution of liver cancer in Serbia is conspicuous and hot spots could be related to drinking water supply. It is very clear that the high-risk regions for PLC occurrence correspond with drinking water reservoirs continually found with cyanobacterial blooms, and the low risk regions correspond with water supplies not affected by cyanobacteria.     

Cyanobacteria Metal Interactions: Requirements,Toxicity, and Ecological Implications

The environmental health-related relevance of cyanobacteria is primarily related to their ability to produce a wide range of toxins, which are known to be hazardous to many organisms, including human beings. The occurrence of cyanobacterial blooms has been related to eutrophic surface water. In the bloom-forming process the levels of phosphorus and nitrogen have been well documented but information regarding concentrations of other chemicals (inorganic, organo-metallic, and organic) is still incipient. Several contaminants, like trace metals, elicit a variety of acute and chronic toxicity effects, but cyanobacteria also have the capability to accumulate, detoxify, or metabolize such substances, to some extent. The role of cyanobacterial exudates has been proved a means of both nutrient acquisition and detoxification. In addition, cyanobacteria are effective biological metal sorbents, representing an important sink for metals in aquatic environment. Understanding the fundamental physicochemical mechanisms of trace metal bio-uptake by cyanobacteria in natural systems is a step towards identifying under what conditions cyanobacterial growth is favored and to ascertain the mechanisms by which blooms (and toxin production) are triggered. In this review the cyanobacterial interactions with metals will be discussed, focusing on freshwater systems.     

LASSBio 596 per os avoids pulmonary and hepatic inflammation induced by microcystin-LR

Cyanobacterial blooms that generate microcystins (MCYSTs) are increasingly recognized as an important health problem in aquatic ecosystems. We have previously reported the impairment of pulmonary structure and function by microcystin-LR (MCYST-LR) exposure as well as the pulmonary improvement by intraperitoneally injected (i.p.) LASSBio 596. In the present study, we aimed to evaluate the usefulness of LASSBio 596 per os on the treatment of pulmonary and hepatic injuries induced by MCYST-LR. Swiss mice received an intraperitoneal injection of 40 μl of saline (CTRL) or a sub-lethal dose of MCYST-LR (40 μg/kg). After 6 h the animals received either saline (TOX and CTRL groups) or LASSBio 596 (50 mg/kg, LASS group) by gavage. Eight hours after the first instillation, lung impedance (static elastance, elastic component of viscoelasticity and resistive, viscoelastic and total pressures) was determined by the end-inflation occlusion method. Left lung and liver were prepared for histology. In lung and hepatic homogenates MCYST-LR, TNF-α, IL-1β and IL-6 were determined by ELISA. LASSBio 596 per os (LASS mice) kept all lung mechanical parameters, polymorphonuclear (PMN) cells, pro-inflammatory mediators, and alveolar collapse similar to control mice (CTRL), whereas in TOX these findings were higher than CTRL. Likewise, liver structural deterioration (hepatocytes inflammation, necrosis and steatosis) and inflammatory process (high levels of pro-inflammatory mediators) were less evident in the LASS than TOX group. LASS and CTRL did not differ in any parameters studied. In conclusion, orally administered LASSBio 596 prevented lung and hepatic inflammation and completely blocked pulmonary functional and morphological changes induced by MCYST-LR.     

A comparative study of Florida strains of Cylindrospermopsis and Aphanizomenon for cylindrospermopsin production.

The toxin cylindrospermopsin (CYN) is produced by a variety of cyanobacterial genera. One of these, Cylindrospermopsis raciborskii, is generally assumed to be the source of CYN in lakes and rivers in Florida, USA. However, in this study, none of the eight Florida isolates of this species tested contained the genetic determinants involved in toxin production nor did they produce CYN. We show for the first time that Aphanizomenon ovalisporum isolated from a pond in this state has the genes putatively associated with CYN production. Analysis by liquid chromatography with mass spectrometric detection (LC/MS) revealed that it produced CYN in the range of 7.39-9.33 microg mg(-1) freeze-dried cells. 16S rDNA sequences of this strain showed 99.6% and 99.9% identity to published A. ovalisporum and Anabaena bergii 16S sequences, respectively. These results help to explain the general lack of a defined relationship between the abundance of C. raciborskii in freshwater ecosystems of Florida and observed concentrations of CYN. The latter observation raises the potential that previous reports of CYN may be coincidental with unrecorded presence of another CYN-producing species.     

Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebrates

Among the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.     

Influence of intracellular toxin concentrations on cylindrospermopsin bioaccumulation in a freshwater gastropod (Melanoides tuberculata).

Scant information is available regarding the bioaccumulation of cylindrospermopsin (CYN) in aquatic organisms, particularly in invertebrates. This study examined toxin bioconcentration and bioaccumulation in the aquatic snail, Melanoides tuberculata, following exposure to freeze-thawed whole cell extracts and a live Cylindrospermopsis raciborskii culture containing CYN. Both bioconcentration and bioaccumulation were evident, but exposure to toxin in the freeze-thawed solutions resulted in minor tissue contamination compared with that resulting from live C. raciborskii exposure. Thus, whilst CYN uptake resulted from both extracellular and intracellular exposures, the availability of intracellular toxin was critical in affecting tissue CYN values. M. tuberculata did not bioconcentrate CYN into the shell. Bioaccumulation of the analog deoxy-CYN was also recorded. Knowledge of intracellular toxin concentrations may be critical in evaluating the bioaccumulation, ecological and human health risks associated with contaminated systems.