No description available.

Thesis (Ph. D.) -- Lincoln University, 2004.

Nature has evolved the ability to form a C-F bond, as exemplified by the bacterium Streptomyces cattleya, which elaborates fluoroacetate (FAc) and 4-fluorothreomne (4- FT). The mechanism of this bond formation are unknown. This thesis probes the biosynthesis of fluoroacetate and 4-fiuorothreonine and in doing so explores the C-F bond forming process. Feeding stable isotope enriched primary metabolites to S. cattleya, followed by (^19)F NMR and GCMS analysis of the resultant fluorometabolites, highlights the role of the glycolytic pathway in delivering a substrate for fluorination. 3-Fluoro-l- hydroxypropan-2-one was synthesised and feeding studies eliminate this as the initial product of fluorination. Fluoroacetaldehyde was identified as a common fluorinated intermediate in the biosynthesis of both FAc and 4-FT. Whole cell studies demonstrate the rapid oxidation of fluoroacetaldehyde to FAc. 4-FT is produced in low quantities by S. cattleya incubated with fluoroacetaldehyde. The synthesis and feeding of [1-(^2)H]- fluoroacetaldehyde provide evidence that the resultant 4-FT is biosynthesised from fluoroacetaldehyde. The biotransformation from fluoroacetaldehyde to FAc was shown in cell free studies to be mediated by an aldehyde dehydrogenase, requiring NAD(^4) as a co-factor. The substrate specificity of fluoroacetaldehyde dehydrogenase was probed by spectrophotometrically monitoring the production of NADH in the presence of different aldehydes. Further cell free experiments probed the biosynthetic origins of fluoroacetaldehyde. Glycolaldehyde phosphate and various phosphorylated glycolytic intermediates were incubated with cell free extracts of S. cattleya and a plethora of co-factors. In the absence of observing fluorination activity, it was shown that the cell free extract acts to dephosphorylate the substrates. The putative role of glycolaldehyde phosphate was explored by feeding isotopically labelled glycolaldehydes to whole cells of the bacterium. The results were not consistent with direct conversion from glycolaldehyde phosphate to fluoroacetaldehyde.

The control of introduced mammalian predators in New Zealand forests is crucial for the protection of native species and essential ecosystem services. Possum control in the form of aerial 1080 applications is conducted by TbFree New Zealand to prevent the spread of bovine tuberculosis, and often has the added conservation benefit of temporarily reducing levels of other mammalian predators such as rodents and mustelids. However, native non-target species such as birds and weta can also be at risk of direct and secondary poisoning following 1080 applications, as well as increased predation risk through mesopredator release. To determine whether the benefits of 1080 applications outweigh the risks to non-target native species, both short and long-term monitoring of populations following aerial 1080 applications is needed. For this study, two forest regions in the South Island were selected for pre- and post-treatment monitoring of non-target species following an aerial 1080 application for possum control. Each region contained a treatment site and a paired non-treatment site. Relative indicies of possums, rodents and other mammalian predators were obtained using tracking tunnels and chew cards, indicies of birds were obtained using five-minute bird counts, and indicies of tree weta were obtained using tracking tunnels and artificial shelters. Monitoring was conducted before the aerial 1080 was applied in August 2012, and over the following 2012/13 and 2013/14 summer seasons. The aerial 1080 applications were successful at reducing possums to undetectable levels at both treatment sites for the two seasons following treatment. Mice were significantly reduced at one treatment site relative to the paired non-treatment site immediately following the 1080 operation, but had increased to pre-1080 levels by the second post-treatment monitoring season. Rats were detected at low levels, and showed no response to the treatment. Mustelids were not detected at either region throughout the monitoring period. No native species showed a decline in a treatment site that was not matched in the non-treatment site. Chaffinches significantly declined at both treatment sites relative to non-treatment sites, likely due to an indirect delayed effect such as competition for food resources. Tomtits showed a positive response to the treatment, significantly increasing in both treatment sites over the post-treatment monitoring periods. Tree weta showed no significant decline in response to the treatment. The reduction of possums to low levels, and the maintenance of possum control with ongoing 1080 operations, is likely to continue to provide an overall net benefit to native non-target species.

Thesis (Ph. D.)--Lincoln University, 2004. / Title from PDF title page (viewed on Oct. 12, 2006). Includes bibliographical references (p. 153-174).

In Western Australia dried meat baits containing 1080 are used extensively by agricultural and conservation organisations to control foxes and dingoes for the protection of agricultural production and native fauna. Field trials were conducted to assess 1080 loss from dried meat baits and this required the analysis of over five hundred baits. Because of this large number of baits it was essential to have a simple and efficient 1080 extraction procedure and method of 1080 analysis. In this study three methods of 1080 extraction and the new bioassay method for 1080 analysis were investigated. A simple and cost-effective 1080 extraction method using water with a 98% 1080 recovery rate was developed and modifications to the bioassay method were made.Factory-produced 1080 dried meat baits were laid in the field during different seasons at four locations in Western Australia, samples were collected over time and analysed for 1080 content using the bioassay. Rainfall was recorded and temperature data was collected for each site. Baits were exposed to the elements but were placed in mesh or wire cages to restrict invertebrate attack and prevent removal by vertebrates. Some baits were placed on the surface and others were buried. Initially 1080 loss from baits from all 4 sites was minimal, ranging from 0 - 21% at day 7 - 9. Further loss was gradual even when rainfall was recorded. Generally baits had to be exposed to at least 50 mm of rain before 1080 loss increased to 50%. At some sites baits continued to remain toxic to foxes even after long exposure. The mean 1080 content of baits from the Carnarvon site at day 226 was 2.0 mg (55% of the mean 1080 content of baits at day zero) with 137 mm of rainfall recorded for that period. Loss of 1080 from baits buried occurred at a faster rate than from baits placed on the surface during the same time period. By day 14 no 1080 was ++ / detected in the buried baits compared to the 68% detected in the surface baits. Under certain conditions 1080 loss from baits was minimal. Levels of 1080 in baits from Nangeen Hill remained fairly constant during the months of September to December 1995, and again during February to April 1996.Gastrolobium plant tissue and soil samples from the southwest of Western Australia were investigated for the presence of 1080 degrading microorganisms. Microbes were isolated and individually tested in solution containing 1080 as the sole carbon source. Isolates which showed 1080 degrading ability were further tested for their degrading efficiency in McClung carbon-free solution with added 1080 as the sole source of carbon and in factory 1080 waste solution, at 1080 concentrations of 20 and 200 mM. The effect of temperature on their rate of degradation was also examined. Thirteen isolates (7 fungi and 6 bacteria) showing varying degrees of 1080 degrading ability were obtained. Rates of 1080 degradation varied among isolates but were highest in the factory waste solution at the 20 mM concentration and in the McClung solution, where 1080 was the sole source of carbon, at the higher concentration of 200 mM. The most efficient isolates OSK and 10H (both Pseudomonas species) degraded all the 1080 present in sterile factory waste solution up to 20 mM 1080 concentration in 4 days and the isolate 1AF (Fusarium oxysporum) degraded 93% of 200 mM 1080 in the McClung solution in 9 days. The optimum temperatures for 1080 degradation were 30 degrees celsius and fluctuating ambient temperatures of 15 28 degrees celsius.

The principal use of sodium monofluoroacetate (1080) in New Zealand is to control brushtail possums (Trichosurus vulpecula). Aerial application of baits containing 1080 is the most common method used for large-scale control of possums. The use of 1080 attracts a great deal of controversy, in particular the effects on the environmental, non-target species, and the potential chronic effects in humans associated with environmental exposures. Although the nature of the acute toxicity of 1080 has been known for more than fifty years, little is known of its effects on humans, in particular its chronic effects to environmental exposures. A benchmark dose (BMD) as an alternative to a no-observed-adverse-effect-level (NOAEL) approach was investigated as a means to improve current health risk assessment values of 1080. Both approaches were investigated for three critical toxicological end points, namely cardiomyopathy, testicular toxicity and teratogenic effects identified from the few available critical studies. The calculated BMDs and lower-bound confidence limits (BMDLs) for the three end points were estimated using the Weibull, probit and quanntal linear models. A benchmark response (BMR) of 10% (extra risk) was chosen and the Akaike�s information criterion (AIC) was used in selecting the appropriate model. The BMDL estimates derived were generally slightly higher but comparable to the corresponding NOAEL for those same endpoints. The computed BMD₁₀ and BMDL₁₀ for cardiomyopathy and testicular effects were 0.21 mg kg⁻�bw⁻� and 0.10 mg kg⁻�bw⁻�, respectively. Tolerable Daily Intakes (TDIs) were derived using the NOAEL approach and the BMD methodology and applying an uncertainty factor of 3000. The resulting TDI using the BMDL were generally consistently slightly higher than those derived using the NOAEL approach. Based on the best fit of modelled dose-response data, a TDI of 0.03 [mu]g kg⁻�bw⁻�day⁻� is proposed for human health risk assessment. Two sets of Provisional Maximum Acceptable Values (PMAV) were derived using the highest concentration of 4.0 [mu]g L⁻� 1080 found in water (N=1450), and using the maximum allowable concentration of 2.0 [mu]g L⁻� of 1080 in water for adults (0.58 [mu]g L⁻� and 0.94 [mu]g L⁻�, respectively) and children (0.23 [mu]g L⁻� and 0.4 [mu]g L⁻�, respectively). Parameters used in the derivation of PMAVs were average weight, average quantity of water consumed, and proportion of total intake allocated to drinking water. The derived adult PMAV of 0.60 [mu]g L⁻� is proposed in revising the PMAV for 1080 in the Drinking Water Standards New Zealand. This value is 6-fold lower than the current PMAV of 3.5 [mu]g L⁻�. Additional toxicology studies are recommended to meet the definition of a "complete database" and therefore estimating a more defensible TDI, and consequently a PMAV for 1080. Risk management approaches are consistent with the Ministry of Health�s current precautionary approach. A PMAV of 0.60 [mu]gL⁻� in drinking water is recommended to consider it suitable for human consumption and that continuous monitoring be carried if the level of 1080 exceeds 50% of the proposed PMAV as a requirement for Priority 2 determinands in the Drinking Water Standards. Precautionary approach appears to be warranted and this was supported by information provided by the Public Health Units (PHU) where 1080 was permitted to be dropped onto drinking water catchments. The PHUs exercised precautionary measures by imposing appropriate conditions to suit local circumstances. As 1080 may likely remain an essential tool to contain tuberculosis spread by possums and to reduce possum damage to forests and crops until better methods of control are developed, a number of recommendations were proposed to protect public health.

The principal use of sodium monofluoroacetate (1080) in New Zealand is to control brushtail possums (Trichosurus vulpecula). Aerial application of baits containing 1080 is the most common method used for large-scale control of possums. The use of 1080 attracts a great deal of controversy, in particular the effects on the environmental, non-target species, and the potential chronic effects in humans associated with environmental exposures. Although the nature of the acute toxicity of 1080 has been known for more than fifty years, little is known of its effects on humans, in particular its chronic effects to environmental exposures. A benchmark dose (BMD) as an alternative to a no-observed-adverse-effect-level (NOAEL) approach was investigated as a means to improve current health risk assessment values of 1080. Both approaches were investigated for three critical toxicological end points, namely cardiomyopathy, testicular toxicity and teratogenic effects identified from the few available critical studies. The calculated BMDs and lower-bound confidence limits (BMDLs) for the three end points were estimated using the Weibull, probit and quanntal linear models. A benchmark response (BMR) of 10% (extra risk) was chosen and the Akaike�s information criterion (AIC) was used in selecting the appropriate model. The BMDL estimates derived were generally slightly higher but comparable to the corresponding NOAEL for those same endpoints. The computed BMD₁₀ and BMDL₁₀ for cardiomyopathy and testicular effects were 0.21 mg kg⁻�bw⁻� and 0.10 mg kg⁻�bw⁻�, respectively. Tolerable Daily Intakes (TDIs) were derived using the NOAEL approach and the BMD methodology and applying an uncertainty factor of 3000. The resulting TDI using the BMDL were generally consistently slightly higher than those derived using the NOAEL approach. Based on the best fit of modelled dose-response data, a TDI of 0.03 [mu]g kg⁻�bw⁻�day⁻� is proposed for human health risk assessment. Two sets of Provisional Maximum Acceptable Values (PMAV) were derived using the highest concentration of 4.0 [mu]g L⁻� 1080 found in water (N=1450), and using the maximum allowable concentration of 2.0 [mu]g L⁻� of 1080 in water for adults (0.58 [mu]g L⁻� and 0.94 [mu]g L⁻�, respectively) and children (0.23 [mu]g L⁻� and 0.4 [mu]g L⁻�, respectively). Parameters used in the derivation of PMAVs were average weight, average quantity of water consumed, and proportion of total intake allocated to drinking water. The derived adult PMAV of 0.60 [mu]g L⁻� is proposed in revising the PMAV for 1080 in the Drinking Water Standards New Zealand. This value is 6-fold lower than the current PMAV of 3.5 [mu]g L⁻�. Additional toxicology studies are recommended to meet the definition of a "complete database" and therefore estimating a more defensible TDI, and consequently a PMAV for 1080. Risk management approaches are consistent with the Ministry of Health�s current precautionary approach. A PMAV of 0.60 [mu]gL⁻� in drinking water is recommended to consider it suitable for human consumption and that continuous monitoring be carried if the level of 1080 exceeds 50% of the proposed PMAV as a requirement for Priority 2 determinands in the Drinking Water Standards. Precautionary approach appears to be warranted and this was supported by information provided by the Public Health Units (PHU) where 1080 was permitted to be dropped onto drinking water catchments. The PHUs exercised precautionary measures by imposing appropriate conditions to suit local circumstances. As 1080 may likely remain an essential tool to contain tuberculosis spread by possums and to reduce possum damage to forests and crops until better methods of control are developed, a number of recommendations were proposed to protect public health.

Enzymatic fluorination of natural products is extremely rare. Of the 4000 halogenated natural products identified, only 13 possess a fluorine atom. The C-F bond forming enzyme from the soil bacterium, Streptomyces cattleya, remains the only native enzyme to be identified that is capable of such biochemistry. It generates 5’-fluoro-5-deoxyadenosine (5‘-FDA) from S-adenosyl-L-methionine (SAM) and F-. The “fluorinase” is the first committed step toward the biosynthesis of the two fluorometabolites, 4-fluorothreonine and fluoroacetate, via the common intermediate, fluoroacetaldehyde (FAld). The enzymatic steps responsible for the conversion of 5’-FDA to the fluorometabolites remained to be fully characterised when this project began. Previously, a purine nucleoside phosphorylase was identified that was capable of generating 5-fluorodeoxyribose-1-phosphate (5-FDRP) from 5’-FDA. 5-FDRP is subsequently isomerised to 5-fluorodeoxyribulose-1-phosphate (5-FDRulP) by an aldose-ketose isomerase enzyme. Chapter 2 describes the identification of the isomerase gene from the genomic DNA of S. cattleya and the corresponding protein product was capable of generating 5-FDRulP from 5-FDRP. The next intermediate, FAld, is generated from 5-FDRulP by a fuculose aldolase. Attempts to identify the aldolase gene from S. cattleya were unsuccessful, however a putative fuculose aldolase from Streptomyces coelicolor was isolated that could generate FAld from 5-FDRulP, which is described in Chapter 3. Following the identification and over expression of a PLP-dependant transaldolase, which generates 4-fluorothreonine (4-FT) from FAld and L-threonine in S. cattleya, Chapter 4 details the successful in vitro reconstitution of fluorometabolite biosynthesis using five over- expressed enzymes. In Chapter 5, attempts to develop a novel assay for fluorinase activity was explored. The colorimetric detection of L-methionine produced by the fluorinase in a coupled L-amino acid oxidase and horseradish peroxidase assay, leading to the oxidation of a dye substance. This was carried out with interest in developing a high-throughput assay for fluorinase mutants, generated by random mutagenesis, in order to identify those with increased activity. In the event, it proved unsuccessful.

O fluoroacetato de sódio (FAS), também conhecido como \"composto 1080\", foi um raticida amplamente utilizado no combate de roedores a partir da década de 40, mas que teve seu uso proibido em diversos países, incluindo o Brasil, devido a sua alta toxicidade e ausência de tratamento efetivo nas intoxicações. A despeito deste fato, diversos casos de intoxicação ainda são registrados em seres humanos e animais, em virtude de seu uso ilícito como raticida doméstico ou adição em iscas nas intoxicações criminosas. O FAS, na natureza, pode ser encontrado, também, em algumas plantas tóxicas de grande importância no Brasil, como a Palicourea marcgravii, e possivelmente seja o princípio ativo tóxico de outras plantas de relevância, como a Mascagnia rigida. Neste sentido, este trabalho objetivou o estudo dos aspectos clínicos e anatomopatológicos encontrados nas intoxicações provocadas pelo FAS, em ratos, comparando estes com os encontrados nas intoxicações por extrato aquoso da M. rígida. Também foram comparadas metodologias analíticas para identificação deste agente tóxico utilizando cromatografia em camada delgada (CCD) e cromatografia de alta eficiência (CLAE) com dois tipos de detectores o de UV e o de Condutividade Iônica. Para tanto, quatro grupos contendo seis ratos Wistar, machos, receberam em dose única por via oral 7,0 mg/kg de FAS ou água destilada ou ainda 1,6 ou 3,2 g/kg de extrato aquoso de M. rígida, sendo avaliados os sinais clínicos apresentados por estes animais. Após eutanásia foram coletados fragmentos de cérebro, coração, fígado, rins, pulmão, intestino e baço para exame histopatológico. Tanto os animais que receberam FAS como os que receberam extrato aquoso de M. rígida apresentaram sintomatologia similar com presença de prostração, desconforto respiratório, tremores, pêlos arrepiados, cromodacriorreia, convulsão tônica e tônico-clônica. Na necropsia destes animais não foram observadas alterações macroscópicas, contudo alterações histopatológicas significativas foram encontradas no cérebro, miocárdio, fígado e rins. Os estudos das análise toxicológicas utilizando as três técnicas cromatográficas mostraram que os resultados obtidos na CCD foram adequados para detecção de FAS nas concentrações acima de 1,0 μg do padrão puro e as análises das duas plantas estudadas apresentaram resultados positivos para FAS. As técnicas utilizando CLAE, com dois diferentes detectores, mostraram que quando se utiliza detector de UV o limite de detecção é de 3,75 μg injetado ou de 75 μg/mL e foi possível, também, detectar a presença de FAS nas duas plantas estudadas, porém a técnica não se apresentou adequada para a detecção de intoxicação em tecido animal. O detector de condutividade iônica mostrou limite de detecção de 0,30 μg/mL, isto é, o mais sensível entre os três métodos estudados, e portanto o mais adequado para diagnóstico de intoxicação em tecido animal; no entanto, para identificação do FAS na M. rigida este método não se mostrou adequado devido a interferentes no extrato aquoso e no soro de animais intoxicados. / Sodium fluoroacetate (SFA), also known as \"compound 1080,\" was a rodenticide widely used in the 1940s, but its use was banned in several countries, including Brazil, for its high toxicity and lack of an effective treatment in poisoning. Despite this fact, several cases of poisoning are still registered in humans and animals, because of their misuse as baits, in addition to criminal poisoning. SFA, in the form of natural molecules, can be found in some toxic plants of great importance in Brazil, such as Palicourea marcgravii, and possibly is the active principle of other plants of relevance, such as Mascagnia rigid. Therefore, this study aimed to evaluate the clinical and pathological findings in poisoning caused by the SFA in rats, and compare them with those found in poisoning by the aqueous extract of M. rigid. Moreover, we compared analytical methods for identifying this toxic agent using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) employing two types of detectors: ultraviolet (UV) and ionic conductivity. Therefore, four groups containing six male Wistar rats each, received a single oral dose of 7.0 mg / kg of SFA, an equal volume of distilled water, or 1.6 or 3.2 g / kg of an aqueous extract of M. rigid, and the clinical signs displayed by these animals were evaluated. After euthanasia fragments of brain, heart, liver, kidney, lung, intestine and spleen were collected for histological examination. Both the animals that received SFA and those who received the aqueous extract of M. rigid showed similar symptoms, including prostration, respiratory distress, tremors, shivering, and tonic or tonic-clonic seizures. At necropsy, these animals did not display macroscopic changes. Nevertheless, significant histopathological changes were found in the brain, heart, liver and kidneys. Toxicological analysis using the three chromatographic techniques described above showed that the results obtained in the TLC were suitable for detection of SFA at concentrations above 1.0 μg of pure standard, and analysis of the two plants mentioned showed positive results for SFA. The techniques using HPLC with two different detectors showed that when using the UV detector, the detection limit is of 3.75 μg (injected) or 75 μg / mL, and SFA could also be detected by these methods in the two plants studied, but the technique does not seem suitable for the detection of intoxication in animal tissue samples. The ionic conductivity detector showed a detection limit of 0.30 μg/ mL, therefore the most sensitive of the three methods studied, and hence is the most appropriate for diagnosis of poisoning in animal tissue; however, for the identification of SFA in the M. rigid, this method was not appropriate due to a putative interference in the aqueous extract.