Development of antidotes for sodium monofluoroacetate (1080)

Baits containing sodium monofluoroacetate (1080) are commonly used in New Zealand during feral pest control operations. However, each year, a number of domestic dogs are unintentionally killed during these control operations, and a suitable antidote to 1080 intoxication is required. The primary toxic mechanism of 1080 is well known. However, as with other pathologies where energy deprivation is the main effect of intoxication, the cascade of effects that arises from this primary mechanism is complex. At present, putative antidotes for 1080 are generally unable to address the primary mechanism of intoxication but such agents may be able to control the cascade of secondary effects, which can result during intoxication. Part of the reason for this is that targeting the cascade can provide a longer window of time for antidote success. We have undertaken studies that identified some of the central nervous system (CNS) and systemic pathophysiological cascades caused by 1080 intoxication. Using this information we designed antidotes, on the basis of preventing different steps in this cascade. In the chicken model targeting systemic changes, in particular reducing effects of nitric oxide derivatives generated in cardiac muscle, proved successful in reducing fatality associated with 1080. In rats and sheep, targeting the CNS with a number of compounds including: glutamate; calcium and dopamine antagonists; gamma amino butyric acid agonists, and astressin-like compounds reduced fatalaties. However, to be successful in the rat and sheep model a given antidote needed to move quickly from systemic circulation across the blood brain barrier and into the CNS. The work also suggests ways in which specific biomarkers of 1080 exposure may be developed with respect to different species.     

The toxicity of sodium monofluoroacetate (1080) to

Although many species of native invertebrates have been identified on toxic baits containing sodium monofluoroacetate (1080) following aerial operations for possum control, few quantitative data are available to determine the risk of primacy or secondary poisoning that may result from these exposures. This paper reports on a series of studies conducted to determine the risk of 1080 exposure to one such non-target insect, the native ant Huberia striata. Subsequent risk of secondary poisoning to insectivorous animals is extrapolated. Ants were exposed in the laboratory to cereal baits containing 0.15% sodium monofluoroacetate, with and without alternative sources of food, and mortality was compared with controls after 24 and 48 h. Acute mortality was significantly greater in both exposed groups and ants that died contained 1080 residues, indicating that some ants could consume lethal amounts of 1080 in cereal baits. However, the increase in mortality was relatively small (7% after 24 h and 12% after 48 h), indicating either that palatability of 1080 bait to this species is low, or that the toxin is not readily absorbed from this matrix. Additional trials were conducted to expose ants to 1080 in sugar-water, in order to determine the acute toxicity dose range. The oral LD50 at 48 h was 32 mg kg(-1), which was comparable to previous results obtained for the large-headed weta, Helmideina crassidens. The persistence of 1080 in ants consuming a sub-lethal dose was also determined, in order to assess risks of secondary poisoning to insectivores. Residues in ants receiving approximately 36 mg kg(-1) of 1080 declined rapidly, from a Peak of 5.51 mg kg(-1) 1 day after exposure, to 0.27 mg kg(-1) after 7 days. Potential risks to insectivorous birds were calculated using worst-case exposure assumptions, and were determined to be negligible.     

Dispersal of an introduced shrub (Crataegus monogyna) by the Brush-tailed Possum (Trichosurus vulpecula)

The Brush-tailed possum Trichosurus vulpecula eats the fleshy fruits of the introduced shrub Crataegus monogyna and defaecates intact seeds at least 0–50 m away. Viability of seeds recovered from faeces was >73%. comparable to the viability of fresh seeds (87.6%). Seed dispersal by possums has been largely neglected and may have important implications for invasion by woody weeds in Australasia.     

Mechlorethamine-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells

Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.     

Relationships Among Families of Diprotodontia (Marsupialia) and the Phylogenetic Position of the Autapomorphic Honey Possum (Tarsipes rostratus)

The Australasian marsupial order Diprotodontia includes ten extant families that are grouped into the suborders Vombatiformes (koalas and wombats), Macropodiformes (kangaroos and allies), and Phalangeriformes (possums and gliders). We investigated interfamilial relationships using mitochondrial 12S rRNA, valine tRNA, and 16S rRNA gene sequences. Our results support the monophyly of both Vombatiformes and Macropodiformes, but not Phalangeriformes. Among possums and gliders, there was strong support for a petauroid clade that includes Pseudocheiridae (ringtail possums), Petauridae (sugar glider, striped possums), Acrobatidae (feathertail possums), and the monotypic family Tarsipedidae, which is represented by the highly specialized and autapomorphic honey possum (Tarsipes rostratus). Other prior hypotheses for the phylogenetic placement of the honey possum were rejected by statistical tests. The inclusion of the honey possum within Petauroidea suggests that derived ultrastructural features of Tarsipes' spermatozoa evolved independently in Tarsipes versus polyprotodont Australasian marsupials.     

Hormonal Correlates of Social Rank in an Asocial Species, the Common Brushtail Possum (Trichosurus vulpecula)

Dominance hierarchies play an important role in access to mates or resources in many species. Rank is sometimes correlated with circulating testosterone levels or morphological traits such as body weight. The relationship of glucocorticoid secretion and rank, however, is less clear. In this study, we investigated the relationship of male rank to body weight, circulating testosterone and cortisol concentrations in captive possum triads (Trichosurus vulpecula). We carried out two experiments to examine hierarchy formation and the effects of castration of the dominant male during the non‐breeding season. A third experiment measured the effects of removal of the dominant male from a stable hierarchy during the breeding season. We found that dominant male rank was significantly correlated with higher circulating testosterone levels during periods of hierarchy formation and during the breeding season but not during periods of hierarchy stability in the non‐breeding season. Lack of correlation between plasma testosterone concentration and rank after male castration suggests that stable social rank is not dependent on hormone level and may be more dependent on behavioural traits. Any biocontrol measures that rely on manipulation of hormone levels may be unreliable when applied to unstable hierarchical situations, including the establishment of territories by subadult males, and the pre‐breeding season when circulating testosterone concentrations peak in males and a period of hierarchy establishment may occur.     

A preliminary report on a bacteriological assay for compound 1080 (sodium monofluoroacetate)

A new, inexpensive, simple and rapid bioassay for sodium monofluoroacetate (compound 1080) was developed using bacteria. Two 1080-sensitive isolates ( Bacillus sp. DHW and Acinetobacter sp. DHW) from local aquatic environments were tested on different agar media using the disc diffusion technique. Both bacteria exhibited zones of growth inhibition surrounding the 1080 discs that were linearly proportional to the concentrations (log₁₀) of 1080. Preliminary studies indicate that this technique can be used for assaying 1080 in environmental and biological samples.     

Fate of sodium monofluroacetate (1080) following disposal of pest bait to a landfill

The results of a programme to monitor the containment and natural breakdown of approximately 12 000 kg of toxic vertebrate pest bait, containing compound 1080 (sodium monofluroacetate), in a landfill site are reported. The baits were buried in a purpose-dug pit in a managed solid waste disposal site at Winton in central Southland, New Zealand, in August 1996. Compound 1080 is used extensively in a bait form to control a range of introduced vertebrate pests, (e.g., European rabbit, Australian brush tailed possum), which cause considerable economic and environmental damage in New Zealand. Two shallow monitor bores, sited 5 and 13 m from the disposal pit, were sampled weekly for five weeks and thereafter monthly for 13 months. Analyses detected 1080 in 5 of the 28 groundwater/leachate samples. The 1080 concentrations in those samples, except for one result, were low. These were either below or close to the Ministry of Health provisional maximum acceptable value standards (PMAV) for drinking water, currently 0.005 mu g ml(-1). The concentrations of 1080 in groundwater in the more distant bore (13 m) were markedly lower than those in the nearer bore (5 m). 1080 was first detected in the near bore after 5 weeks and the more distant bore after 16 weeks. The level and frequency of incidence of 1080 in both holes decreased over the sampling period until none was detected after 10 months. In situ sampling of the residual waste material indicated the 1080 concentration in the disposal pit decreased to less than 10% of its original level in 12 months. The active anaerobic bacterial processes operating in the organic refuse pile appear to provide an ideal environment for the rapid natural breakdown of 1080. The findings will assist with the setting of conditions for resource consents concerning the disposal of materials containing 1080 in landfill sites.     

Variant Association Tools for Quality Control and Analysis of Large-Scale Sequence and Genotyping Array Data

Currently there is great interest in detecting associations between complex traits and rare variants. In this report, we describe Variant Association Tools (VAT) and the VAT pipeline, which implements best practices for rare-variant association studies. Highlights of VAT include variant-site and call-level quality control (QC), summary statistics, phenotype- and genotype-based sample selection, variant annotation, selection of variants for association analysis, and a collection of rare-variant association methods for analyzing qualitative and quantitative traits. The association testing framework for VAT is regression based, which readily allows for flexible construction of association models with multiple covariates and weighting themes based on allele frequencies or predicted functionality. Additionally, pathway analyses, conditional analyses, and analyses of gene-gene and gene-environment interactions can be performed. VAT is capable of rapidly scanning through data by using multi-process computation, adaptive permutation, and simultaneously conducting association analysis via multiple methods. Results are available in text or graphic file formats and additionally can be output to relational databases for further annotation and filtering. An interface to R language also facilitates user implementation of novel association methods. The VAT''s data QC and association-analysis pipeline can be applied to sequence, imputed, and genotyping array, e.g., “exome chip,” data, providing a reliable and reproducible computational environment in which to analyze small- to large-scale studies with data from the latest genotyping and sequencing technologies. Application of the VAT pipeline is demonstrated through analysis of data from the 1000 Genomes project.     

Purification and Characterisation of Hepatic Glutathione Transferases from a Herbivorous Marsupial, the Brushtail Possum (Trichosurus vulpecula)

A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 ± 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1–1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1–1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1–1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1–1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1–1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.     

Large-Scale Preparation of Biologically Active Recombinant Chicken Obese Protein (Leptin)

Prokaryotic expression vector pMON3401 encoding full size A(−1) chicken leptin (AF012727) was prepared by PCR of previously described cDNA.Escherichia colicells transformed with this vector overexpressed large amounts of chicken leptin upon induction with nalidixic acid. The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on a Q-Sepharose column, yielding two electrophoretically pure fractions (leptin-1 and leptin-2), eluted from the column by 100 and 125 mM NaCl. Both fractions showed a single band of the expected molecular mass of 16 kDa and were composed of over 95% of monomeric protein. The biological activity of both fractions, resulting from proper renaturation, was further evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin–receptor construct and by lowering the food intake of starved chicken following intravenal or intraperitoneal injections.     

The impact of 1080 bait removal by Lace Monitors (Varanus varius) on a Red Fox (Vulpes vulpes) control programme

The use of toxic bait over large areas is a cost‐effective and widely used technique for the management of wild canids in Australia. Lace Monitors are known to also take baits, and though unlikely to be lethal to these reptiles the effect on the efficacy of control operations is largely unknown. During a five year Red Fox control programme in eastern Victoria, Lace Monitors removed significantly higher numbers of toxic baits than canids during the warmer months, reducing the likelihood of foxes encountering bait and reducing the efficacy of the control programme. Suggestions are made to mitigate bait take by Lace Monitors.     

Bacterioplankton Community Structure and Dynamics after Large-Scale Release of Nonindigenous Bacteria as Revealed by Low-Molecular-Weight-RNA Analysis

[This corrects the article on p. 3388 in vol. 58.].