More than just an eagle killer: The freshwater cyanobacterium Aetokthonos hydrillicola produces highly toxic dolastatin derivatives.

Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the "eagle killer" neurotoxin aetokthonotoxin (AETX) causing the fatal neurological disease vacuolar myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine-derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode Caenorhabditis elegans. Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product.

Di-N-octylphthalate acts as a proliferative agent in murine cell hepatocytes by regulating the levels of TGF-ß and pro-apoptotic proteins.

Di-n-octylphthalate (DNOP) is a phthalate used in the manufacturing of a wide variety of polyvinyl chloride-containing medical and consumer products. A study on chronic exposure to DNOP in rodents showed the development of pre-neoplastic hepatic lesions following exposure to a tumor initiator. The objective of this study was to identify the mechanisms by which DNOP leads to pre-neoplastic hepatic lesions. Mouse hepatocyte AML-12 and FL83B cells were treated with DNOP. The rate of cell proliferation was increased in treated cells in a concentration-dependent manner. DNOP increased the expression of transforming growth factor-ß (tgf-ß) in both cell lines, and primary culture mouse hepatocytes. The TGF-ß receptor inhibitor LY2109761 impaired the effect of DNOP. The presence of pro-apoptotic proteins decreased in the presence of DNOP. Our observation indicates that DNOP, through an increase in the expression of tgf-ß and a decrease in the levels of pro-apoptotic proteins, acts as a proliferative agent in normal mouse hepatocytes. We also studied the morphological and functional changes of the mouse liver upon a short-term treatment of DNOP. Mice exposed to DNOP displayed an epithelial-to-mesenchymal transition and cholestasis, which was reflected in an increase in hepatic bile acids and glutathione levels.

An extract of Hydrilla verticillata and associated epiphytes induces avian vacuolar myelinopathy in laboratory mallards.

Avian vacuolar myelinopathy (AVM) is a neurological disease affecting bald eagles (Haliaeetus leucocephalus), American coots (Fulica americana), waterfowl, and other birds in the southeastern United States. The cause of the disease is unknown, but is thought to be a naturally produced toxin. AVM is associated with aquatic macrophytes, most frequently hydrilla (Hydrilla verticillata), and researchers have linked the disease to an epiphytic cyanobacterial species associated with the macrophytes. The goal of this study was to develop an extraction protocol for separating the putative toxin from a hydrilla-cyanobacterial matrix. Hydrilla samples were collected from an AVM-affected reservoir (J. Strom Thurmond Lake, SC) and confirmed to contain the etiologic agent by mallard (Anas platyrhynchos) bioassay. These samples were then extracted using a solvent series of increasing polarity: hexanes, acetone, and methanol. Control hydrilla samples from a reference reservoir with no history of AVM (Lake Marion, SC) were extracted in parallel. Resulting extracts were administered to mallards by oral gavage. Our findings indicate that the methanol extracts of hydrilla collected from the AVM-affected site induced the disease in laboratory mallards. This study provides the first data documenting for an "extractable" AVM-inducing agent.

Investigation of the link between avian vacuolar myelinopathy and a novel species of cyanobacteria through laboratory feeding trials.

Avian vacuolar myelinopathy (AVM) is a neurologic disease affecting Bald Eagles (Haliaeetus leucocephalus), American Coots (Fulica americana), and other birds in the southeastern United States. The cause of the disease has not yet been determined, although it is generally thought to be a natural toxin. Previous studies have linked AVM to aquatic vegetation, and the current working hypothesis is that a species of cyanobacteria growing epiphytically on that vegetation is producing a toxin that causes AVM. Surveys of epiphytic communities have identified a novel species of cyanobacteria in the order Stigonematales as the most likely suspect. The purpose of this study was to further examine the relationship between the suspect Stigonematales species and induction of AVM, by using animal feeding trials. Adult Mallards and domestic chickens were fed aquatic vegetation from two study sites containing the suspect cyanobacterial epiphyte, as well as a control site that did not contain the Stigonematales species. Two trials were conducted. The first trial used vegetation collected during mid-October 2003, and the second trial used vegetation collected during November and December 2003. Neither treatment nor control birds in the first trial developed AVM lesions. Ten of 12 treatment Mallards in the second trial were diagnosed with AVM, and control birds were not affected. This study provides further evidence that the novel Stigonematales species may be involved with AVM induction, or at the least it is a good predictor of AVM toxin presence in a system. The results also demonstrate the seasonal nature of AVM events.

Activity patterns of biotransformation enzymes in juvenile chickens after in ovo dosage of PCB126.

Little is known about the correlations between biotransformation enzymes in juvenile birds after exposure to environmental toxicants like PCBs. In this study eggs of domestic chicken (Gallus domesticus) were dosed with PCB126 in concentrations of 0.175-0.325 ng/g egg weight. Liver subcellular fractions were analyzed for activities of Phase 1 and Phase 2 biotransformation enzymes 2 and 5 weeks post-hatch. Ethoxyresorufin O-deethylase (EROD) activity was increased in both the 2-week and 5-week samples. Glutathione-S-transferase (GST) activity was increased in the 2-week samples only, but the 5-week samples showed an overall much higher GST activity, probably as a result of a still developing enzyme expression in maturing chickens. The same pattern was seen in the phenol-type UDP-glucuronosyltransferase (UGT) activity of the control animals. The week two samples showed a positive dose-response relationship for the UGT activity, but after 5 weeks this was reversed, possibly caused by inhibition of hydroxylated PCB metabolites. Phenol-type sulfotransferase (SULT) activities were not significantly correlated with time or dose. There was a strong positive regression between the Ah-receptor mediated EROD and UGT activities. The EROD activities were also positively correlated to the GST activities. Most interesting was a negative correlation between the UGT and SULT activities: an inhibited UGT activity appeared to be compensated by an increased SULT activity.

Concentrations of p,p'-DDE in plasma of nestling Wood Storks from Georgia.

We determined the concentrations of organochlorine pesticides and total PCBs in plasma of nestling Wood Storks (Mycteria americana) from two colonies in Georgia in 2000. Of 20 compounds analyzed for, only p,p'-DDE was quantified in reportable concentrations, ranging from <2.0-174.0 ng/g wet weight (ww). Concentrations of p,p'-DDE were significantly greater (P<0.0001) in plasma of nestlings from an inland colony, Chew Mill (x 16.9 ng/g,ww) than a coastal colony, Harris Neck (x 1.2 ng/g,ww). Concentrations from nestlings from Chew Mill were significantly different among sampling weeks (P=0.01), with week 8 being significantly greater than weeks 1, 2, and 5. While the Chew Mill colony had much greater concentrations of p,p'-DDE in plasma of nestlings than the Harris Neck colony, reproduction was greater there (1.8+/-1.0 SD fledged young per nesting attempt) than Harris Neck (1.4+/-1.0 SD fledged young per nesting attempt). While concentrations of p,p'-DDE determined in plasma of nestling Wood Storks are an indirect measure of adult exposure to environmental toxicants, concentrations reported would not be considered detrimental to reproduction in these colonies.

Avian vacuolar myelinopathy linked to exotic aquatic plants and a novel cyanobacterial species.

Invasions of exotic species have created environmental havoc through competition and displacement of native plants and animals. The introduction of hydrilla (Hydrilla verticillata) into the United States in the 1960s has been detrimental to navigation, power generation, water intake, and water quality (McCann et al., 1996). Our field surveys and feeding studies have now implicated exotic hydrilla and associated epiphytic cyanobacterial species as a link to avian vacuolar myelinopathy (AVM), an emerging avian disease affecting herbivorous waterbirds and their avian predators. AVM, first reported in 1994, has caused the death of at least 100 bald eagles (Haliaeetus leucocephalus) and thousands of American coots (Fulica americana) at 11 sites from Texas to North Carolina (Thomas et al., 1998; Rocke et al., 2002). Our working hypothesis is that the agent of this disease is an uncharacterized neurotoxin produced by a novel cyanobacterial epiphyte of the order Stigonematales. This undescribed species covers up to 95% of the surface area of leaves in reservoirs where bird deaths have occurred from the disease. In addition, this species is rare or not found on hydrilla collected at sites where AVM disease has not been diagnosed. Laboratory feeding trials and a sentinel bird study using naturally occurring blooms of cyanobacteria on hydrilla leaves and farm-raised mallard ducks (Anas platyrhynchos) induced the disease experimentally. Since 1994 AVM has been diagnosed in additional sites from Texas to North Carolina. Specific site characteristics that produce the disjunct distribution of AVM are unknown, but it is probable that the incidence of this disease will increase with the introduction of hydrilla and associated cyanobacterial species into additional ponds, lakes, and reservoirs.